U.S. patent application number 17/091247 was filed with the patent office on 2021-05-20 for inhibitors of nhr2 and/or runx1/eto-tetramerization.
The applicant listed for this patent is Heinrich-Heine-Universitat Dusseldorf. Invention is credited to Holger Gohlke, Manuel Grez, Alexander Metz, Julia Schanda, Christian Wichmann.
Application Number | 20210147415 17/091247 |
Document ID | / |
Family ID | 1000005358595 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210147415 |
Kind Code |
A1 |
Metz; Alexander ; et
al. |
May 20, 2021 |
INHIBITORS OF NHR2 AND/OR RUNX1/ETO-TETRAMERIZATION
Abstract
The invention relates to inhibitors of the NHR2 tetramerization
and their use as tumor therapeutics (e.g. against acute myeloid
leukemia (AML)), cytostatics, and diagnostic agents.
Inventors: |
Metz; Alexander; (Niddatal,
DE) ; Gohlke; Holger; (Dusseldorf, DE) ;
Schanda; Julia; (Berlin, DE) ; Wichmann;
Christian; (Planegg, DE) ; Grez; Manuel;
(Hamburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Heinrich-Heine-Universitat Dusseldorf |
Dusseldorf |
|
DE |
|
|
Family ID: |
1000005358595 |
Appl. No.: |
17/091247 |
Filed: |
November 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15837235 |
Dec 11, 2017 |
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17091247 |
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14787151 |
Oct 26, 2015 |
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PCT/EP2014/058449 |
Apr 25, 2014 |
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15837235 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5011 20130101;
C07D 235/28 20130101; G01N 2333/47 20130101; C07C 59/68 20130101;
C07D 493/04 20130101; C07D 239/22 20130101; C07D 207/337 20130101;
C07D 471/04 20130101; C07D 209/42 20130101; C07C 59/70 20130101;
G01N 27/447 20130101; C07D 317/60 20130101; C07D 307/68
20130101 |
International
Class: |
C07D 471/04 20060101
C07D471/04; C07D 317/60 20060101 C07D317/60; C07D 235/28 20060101
C07D235/28; C07D 493/04 20060101 C07D493/04; C07D 207/337 20060101
C07D207/337; C07D 209/42 20060101 C07D209/42; C07D 307/68 20060101
C07D307/68; C07C 59/70 20060101 C07C059/70; C07C 59/68 20060101
C07C059/68; C07D 239/22 20060101 C07D239/22; G01N 27/447 20060101
G01N027/447; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 2013 |
EP |
13165993.0 |
Claims
1. A method of treatment comprising: treating leukemia with a
compound according to general formula (A), ##STR00019## wherein
R.sub.A0 is --H or --F; --R.sub.A1 and R.sub.A2, respectively
independently, are --H, --F, or --OC.sub.1-8-alkyl; R.sub.A3,
R.sub.A4, and R.sub.A5, respectively independently, are --H, --F,
or --OC.sub.1-8-alkyl; or R.sub.A3 is --H or --OC.sub.1-8-alkyl;
and R.sub.A4 and R.sub.A5 are vicinal and together with the carbon
atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(C.sub.1-8-alkyl)--O--; R.sub.A6 is
--H or --C.sub.1-8-alkyl; R.sub.A7 is --C(.dbd.O)OH or
--C(.dbd.O)OR.sub.S; and m.sub.A means 0, 1, or 2; or a
physiologically acceptable salt thereof; wherein R.sub.X,
respectively independently, is --C.sub.1-8-aliphatic,
--C.sub.3-12-cycloaliphatic, -aryl, heteroaryl,
--C.sub.1-8-aliphatic--C.sub.3-12-cycloaliphatic,
--C.sub.1-8-aliphatic-aryl, --C.sub.1-8-aliphatic-heteroaryl,
--C.sub.3-8-cycloaliphatic--C.sub.1-8-aliphatic,
--C.sub.3-8-cycloaliphatic-aryl, or
--C.sub.3-8-cycloaliphatic-heteroaryl; wherein "aliphatic",
respectively independently, is a branched or unbranched, saturated
or a mono- or polyunsaturated, unsubstituted or mono- or
polysubstituted, aliphatic hydrocarbon residue; wherein in each
case "cycloaliphatic", respectively independently, is a saturated
or a mono- or polyunsaturated, unsubstituted or mono- or
polysubstituted, alicyclic, mono- or multicyclic hydrocarbon
residue; wherein with respect to "aliphatic" and "cycloaliphatic",
"mono- or polysubstituted", respectively independently, is the
mono- or polysubstitution of one or more hydrogen atoms by --F,
--Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2-R.sub.X, --S(--O).sub.1-2NH.sub.2, --NH.sub.2,
--NHR.sub.X, --N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; wherein "aryl",
respectively independently, is a carbocyclic ring system with at
least one aromatic ring, but without heteroatoms in this ring,
wherein, if necessary, the aryl residues can be condensed with
further saturated, (partially) unsaturated or aromatic ring
systems, and each aryl residue can be present in unsubstituted or
mono- or polysubstituted form, wherein the aryl substituents can be
the same or different and in any desired and possible position of
the aryl; wherein "heteroaryl", respectively independently, is a
5-, 6- or 7-membered cyclic aromatic residue, which contains 1, 2,
3, 4 or 5 heteroatoms, wherein the heteroatoms, the same or
different, are nitrogen, oxygen or sulphur, and the heterocycle can
be unsubstituted or mono- or polysubstituted; wherein in the case
of the substitution on the heterocycle the substituents can be the
same or different and can be in any desired and possible position
of the heteroaryl; and wherein the heterocycle can also be part of
a bi- or polycyclic system; wherein with respect to "aryl" and
"heteroaryl", "mono- or polysubstituted", respectively
independently, is the mono- or polysubstitution of one or more
hydrogen atoms of the ring system by substituents selected from the
group comprising --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO,
.dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)--N(R.sub.X).sub.2, --OH, --O(CH.sub.2).sub.1-2O--,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X,
--NH--C(.dbd.O)NH.sub.2, --NHC(.dbd.O)NHR.sub.X,
--NHC(.dbd.O)--N(R.sub.X).sub.2, --Si(R.sub.X).sub.3 and
--PO(OR.sub.X).sub.2; wherein if necessary N-ring atoms present can
be respectively oxidized.
2. The method according to claim 1, wherein R.sub.A1 and R.sub.A2,
respectively independently, are --H or --OC.sub.1-8-aliphatic; or
R.sub.A0 is --H; and R.sub.A1 and R.sub.A2 are vicinal and together
with the carbon atoms to which they are attached form a ring and
mean --O--CH.sub.2--O-- or --O--CH(C.sub.1-8-aliphatic)--O--;
R.sub.A3, R.sub.A4, and R.sub.A5, respectively independently, are
--H or --OC.sub.1-8-aliphatic; or R.sub.A3 is --H or
--OC.sub.1-8-aliphatic; and R.sub.A4 and R.sub.A5 are vicinal and
together with the carbon atoms to which they are attached form a
ring and are --O--CH.sub.2--O-- or
--O--CH(C.sub.1-8-aliphatic)--O--; and R.sub.A6 is H or
--C.sub.1-8-aliphatic.
3. The method according to claim 1, wherein R.sub.A0 is H; R.sub.A1
and R.sub.A2, respectively independently, are --OC.sub.1-8-alkyl;
or R.sub.A1 and R.sub.A2 are vicinal and together with the carbon
atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(C.sub.1-8-alkyl)--O--; and R.sub.A3,
R.sub.A4, and R.sub.A5, respectively independently, are --H or
--OC.sub.1-8-alkyl; or R.sub.A3 is H or --OC.sub.1-8-alkyl; and
R.sub.A4 and R.sub.A5 are vicinal and together with the carbon
atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(C.sub.1-8-alkyl)--O--.
4. The method according to claim 1 wherein R.sub.A0 is H; R.sub.A1
and R.sub.A2, respectively independently, are --OCH.sub.3; or
R.sub.A1 and R.sub.A2 are vicinal and together with the carbon
atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(CH.sub.3)--O--; R.sub.A3, R.sub.A4,
and R.sub.A5, respectively independently, are --H or --OCH.sub.3;
or R.sub.A3 means --H; and R.sub.A4 and R.sub.A5 are vicinal and
together with the carbon atoms to which they are attached form a
ring and are --O--CH.sub.2--O-- or --O--CH(CH.sub.3)--O--; R.sub.A6
is H or --CH.sub.3; R.sub.A7 is --CO.sub.2H; and M.sub.A is 0 or
1.
5. The method according to claim 1, wherein the compound is:
2,4-di(benzo[d][1,3]dioxol-5-yl)-4-oxobutanoic acid having a
structure: ##STR00020## or the physiologically acceptable salts
and/or prodrugs thereof.
6. (canceled)
7. (canceled)
8. (canceled)
9. The method according to claim 1, wherein the leukemia is myeloid
leukemia.
10. The method according to claim 9, wherein the myeloid leukemia
is selected from the group consisting of acute myeloid leukemia,
promyeloid leukemia, acute promyeloid leukemia, promyelocytic
leukemia, acute promyelocytic leukemia, megakaryoblastic leukemia
and acute megakaryoblastic leukemia.
11. The method of claim 1, wherein the treating leukemia with the
compound includes administering the compound at least once daily,
to a subject in need thereof.
12. The method of claim 1, wherein the treating leukemia with the
compound includes administering the compound (i) systemically,
locally or extracorporeally; and/or (ii) orally or
parenterally.
13. The method of claim 1, wherein the method comprises measuring
the inhibition in a subject to which the compound is administered
of NHR2 and/or RUNX1/ETO-tetramerization in an assay selected from
the group consisting of ABCD, EMSA, ELISA and cross-linking assay
and others.
14. The method of claim 1, where the compound is used as a
searcher, biotechnological tool or in diagnostics.
15. The method of claim 14, comprising using the compound in assays
for the diagnosis of diseases associated with the tetramerization
of NHR2 and/or NHR2 containing proteins; in assays for screening
for the tetramerization of NHR2 and/or of NHR2 containing proteins
in vitro, ex vivo, in vivo, in mammals and humans, and the like; or
as a reference compound or competing binder/inhibitor with respect
to inhibition of NHR2 and/or RUNX1/ETO-tetramerization.
16. (canceled)
17. (canceled)
18. (canceled)
Description
FIELD
[0001] The invention relates to inhibitors of the NHR2
tetramerization from dimers and their use as tumor therapeutics
(e.g. against acute myeloid leukemia (AML)), cytostatics, and
diagnostic agents.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The contents of the electronic sequence listing
(SequenceListingProjectFile_1873_001USC1D1_ST25,txt: Size: 3
Kilobytes; and Date of Creation: Feb. 1, 2021) are herein
incorporated by reference in its entirety.
BACKGROUND
[0003] Chromosomal translocations are frequent events during
malignant cell transformation, particularly during leukemogenesis.
The translocation t(8:21) is one of the most frequent chromosomal
anomalies in acute myeloid leukemia. It involves the RUNX1 gene on
chromosome 21 and the ETO gene on chromosome 8. RUNX1 is a
transcription factor and belongs to the key regulators of
hematopoietic cell differentiation.
[0004] The fusion protein RUNX1/ETO has a modular structure.
RUNX1/ETO as such is a monomer but forms RUNX1/ETO dimers and
RUNX1/ETO tetramers. RUNX1/ETO contains four functional domains,
which are generally referred to as nervy homology regions (NHR1 to
NHR4).
[0005] Several in vitro and in vivo studies have shown that the
nervy homology region 2 (NHR2) is essential for RUNX1/ETO oncogenic
activity. The NHR2 domain mediates tetramer formation through
hydrophobic and ionic/polar interactions. Two RUNX1/ETO dimers are
subsequently positioned on top of each other in a sandwich-like
fashion. Similar to RUNX1/ETO monomers, RUNX1/ETO dimers fail to
bind efficiently to DNA and to alter expression of RUNX1-dependent
genes. RUNX1/ETO dimers do not block myeloid differentiation, are
unable to enhance the self-renewal capacity of hematopoietic
progenitors, and fail to induce leukemia in a murine
transplantation model. The crystal structure of the
homo-tetramerization domain of ETO, i.e. the nervy homology region
2 (NHR2), is known from the prior art. In this context, reference
can be made, for example, to Y. Liu et at., Cancer Cell, 2006, 9,
249-260, and C. Wichmann et al., Blood, 2010, 116(4), 602-613.
SUMMARY
[0006] It was an object of the invention to provide compounds that
are capable of inhibiting NHR2 tetramerization and/or
RUNX1/ETO-tetramerization or the tetramerization of other
NHR2-containing proteins that include, but are not limited to, wild
type ETO proteins, ETO homologs or the CBFA2T3-GLIS2 fusion
proteins, preferably without affecting dimer formation, it was
another object of the invention to provide compounds that are
useful in the treatment of diseases and disorders that are
associated with NHR2 and/or RUNX1/ETO-tetramerization. Further, it
was an object of the invention to provide compounds useful as
searcher, biotechnological tool or in diagnostics related to NHR2
and/or RUNX1/ETO-tetramerization.
[0007] This object has been achieved by the subject-matter of the
patent claims
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A, 1B, and 1C illustrate the principle of biochemical
assays for measuring inhibition of NHR2 tetramerization.
[0009] FIGS. 2A, 2B, 2C, 2D, 2E, and 2F illustrate the inhibition
of NHR2 tetramerization.
[0010] FIGS. 3A, 3B, and 3C illustrate a cell survical assay
measuring the effect of inhibition of NHR2 tetramerization on
cells.
[0011] FIGS. 4A and 4B illustrate sequences of the peptides used in
NHR2 inhibition assays aligned to the wild type NHR2 sequence and
protein constructs used in ELISA and ABCD assays.
[0012] FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, and H illustrate reduced
tumor growth of SKNO-1 cells in SCID-gc mice after treatment.
DETAILED DESCRIPTION
[0013] It has been found that the compounds according to the
invention may act as inhibitors of the NHR2 and/or
RUNX1/ETO-tetramerization, which is associated with different
diseases, such as leukemia, particularly acute myeloid
leukemia.
[0014] Further, it has been found that the compounds according to
the invention inhibit tetramer formation without affecting dimer
formation.
[0015] A first aspect of the invention relates to a compound
according to general formula (A),
##STR00001##
wherein [0016] R.sub.A0, R.sub.A1 and R.sub.A2, respectively
independently, mean --H, --F, --Cl, --Br, --I, --CN, --NO.sub.2,
--CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H,
--C(.dbd.O)OH, --C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, --C(O)N(R.sub.X).sub.2, --OH, --OR.sub.X,
--OC(.dbd.O)H, --OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X,
--OC(.dbd.O)NHR.sub.N, --OC(.dbd.O)N(R.sub.X).sub.2, --SH,
--SR.sub.X, --SO.sub.3H, --S(.dbd.O).sub.1-2--R.sub.X,
--S(.dbd.O).sub.1-2NH.sub.2, --NH.sub.2, --NHR.sub.X,
--N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0017] or R.sub.A0
means --H, --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NHR.sub.X, --N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; and R.sub.A1 and
R.sub.A2 are vicinal and together with the carbon atoms to which
they are attached form a ring and mean --O--CH.sub.2--O-- or
--O--CH(R.sub.X)--O-- or --O--C(R.sub.X).sub.2--O--; [0018]
R.sub.A3, R.sub.A4, and R.sub.A5, respectively independently, mean
--H, --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O --,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0019] or R.sub.A3
means --H, --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S('O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2, --NH.sub.2,
--NHR.sub.X, --N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; and R.sub.A4and
R.sub.A5are vicinal and together with the carbon atoms to which
they are attached form a ring and mean --O--CH.sub.2--O-- or
--O--CH(R.sub.X)--O--, [0020] R.sub.A6 means --H, --F, --Cl, --Br,
--CN, --CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H,
--C(.dbd.O)OH, --C(.dbd.O)OR.sub.X, --C(O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, --O(.dbd.O)N(R.sub.X).sub.2, --OH,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0021] R.sub.A7 means
--F, --Cl, --Br, --CN, --NO.sub.2, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)OH, --C(.dbd.O)OR.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X),
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0022] m.sub.A means
0, 1, 2, 3, 4, 5 or 6; [0023] or a physiologically acceptable salt
and/or a prodrug thereof.
[0024] In a preferred embodiment of the compound according to
general formula (A),
[0025] R.sub.A0, R.sub.A1, and R.sub.A2, respectively
independently, mean --H or --OC.sub.1-8-aliphatic; [0026] or
R.sub.A0 means --H or --OC.sub.1-8-aliphatic; and R.sub.A1 and
R.sub.A2 are vicinal and together with the carbon atoms to which
they are attached form a ring and mean --O--CH.sub.2--O-- or
--O--CH(C.sub.1-8-aliphatic)--O--; [0027] R.sub.A3, R.sub.A4, and
R.sub.A5, respectively independently, mean --H or
--OC.sub.1-8-aliphatic: [0028] or R.sub.A3 means --H or
--OC.sub.1-8-aliphatic; and R.sub.A4 and R.sub.A5 are vicinal and
together with the carbon atoms to which they are attached form a
ring and mean or --O--CH.sub.2--O-- or
--O--CH(C.sub.1-8-aliphatic)--O--; [0029] R.sub.A6 means --H or
--C.sub.1-8-aliphatic; [0030] R.sub.A7 means --C(.dbd.O)R.sub.X,
--C(.dbd.O)H, --C(.dbd.O)OH, --C(.dbd.O)OR.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X, or
--C(.dbd.O)N(R.sub.X).sub.2 and [0031] m.sub.A means 0, 1, 2, 3, or
4.
[0032] In another preferred embodiment of the compound according to
general formula (A), [0033] R.sub.A0 means --H; [0034] R.sub.A1 and
R.sub.A2, respectively independently, mean --OC.sub.1-8-alkyl;
[0035] or R.sub.A1 and R.sub.A2 are vicinal and together with the
carbon atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(C.sub.1-6-alkyl)--O--: [0036]
R.sub.A3, R.sub.A4, and R.sub.A5, respectively independently, mean
--H or --OC.sub.1-8-alkyl; [0037] or R.sub.A3 means --H or
--OC.sub.1-8-alkyl; and R.sub.A4 and R.sub.A5 are vicinal and
together with the carbon atoms to which they are attached form a
ring and mean --O--CH.sub.2--O-- or --O--CH(C.sub.1-8-alkyl)--O--,
[0038] R.sub.A6 means --H or --C.sub.1-8-alkyl; [0039] R.sub.A7
means --C(.dbd.O)OH or --C(.dbd.O)OR.sub.X; and [0040] m.sub.A
means 0, 1, or 2.
[0041] In a further preferred embodiment according to general
formula (A), [0042] R.sub.A0 means --H; [0043] R.sub.A1 and
R.sub.A2, respectively independently, mean --OCH.sub.3, [0044] or
R.sub.A1 and R.sub.A2 are vicinal and together with the carbon
atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(CH.sub.3)--O--;
[0045] R.sub.A3, R.sub.A4, and R.sub.A5, respectively
independently, mean --H or --OCH.sub.3; [0046] or R.sub.A3 means
--H: and R.sub.A4 and R.sub.A5 are vicinal and together with the
carbon atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(CH.sub.3)--O--; [0047] R.sub.A6 means
--H or --CH.sub.3; [0048] R.sub.A7 means --CO.sub.2H; and [0049]
m.sub.A means 0 or 1.
[0050] Particularly preferred embodiments of the compound according
to general formula (A) have the general formulas (A-1) or
(A-2):
##STR00002##
[0051] Compounds of the general formula (A-1) that particularly
preferred are those, wherein [0052] R.sub.A0 means --H; [0053]
R.sub.A1 and R.sub.A2 together with the carbon atoms to which they
are attached form a ring and mean --O--CH(CH.sub.3)--O--.
[0054] R.sub.A3, R.sub.A4, and R.sub.A5, respectively
independently, mean --H or --OCH.sub.3, [0055] or R.sub.A3 means
--H; and R.sub.A4 and R.sub.A5 and together with the carbon atoms
to which they are attached form a ring and mean --O--CH.sub.2--O--
or --O--CH(CH.sub.3)--O--; [0056] R.sub.A6 means --H or --CH.sub.3;
[0057] R.sub.A7 means --C(.dbd.O)OH or --C(.dbd.O)OR.sub.X; and
[0058] m.sub.A means 0 or 1.
[0059] Compounds of the general formula (A-1) that are more
particularly preferred are those, wherein [0060] R.sub.A0 means
--H; [0061] R.sub.A1 and R.sub.A2together with the carbon atoms to
which they are attached form a ring and mean
--O--CH(CH.sub.3)--O--; [0062] R.sub.A3 means --H, and R.sub.A4 and
R.sub.A5 together with the carbon atoms to which they are attached
form a ring and mean --O--CH.sub.2--O--. [0063] R.sub.A6 means H;
[0064] R.sub.A7 means --CO.sub.2H; [0065] m.sub.A means 0.
[0066] Compounds of the general formula (A-2) that are particularly
preferred are those, wherein [0067] R.sub.A3, R.sub.A4, and
R.sub.A5, respectively independently, mean --H or --OCH.sub.3;
[0068] or R.sub.A3 means --H; and R.sub.A4 and R.sub.A5 together
with the carbon atoms to which they are attached form a ring and
mean --O--CH.sub.2--O-- or --O--CH(CH.sub.3)--O--; [0069] R.sub.A6
means --H or --CH.sub.3; [0070] m.sub.A means 0 or 1.
[0071] Compounds of the general formula (A-2) that are more
particularly preferred are those, wherein [0072] R.sub.A3,
R.sub.A4, and R.sub.A5, respectively independently, mean
--OCH.sub.3; [0073] or R.sub.A3 means --H; and R.sub.A4, and
R.sub.A5 together with the carbon atoms to which they are attached
form a ring and mean --O--CH.sub.2--O--;
[0074] R.sub.A6 means --H or --CH.sub.3; [0075] m.sub.A means 0 or
1.
[0076] Compounds from the following group are most particularly
preferred: [0077] 2,4-cli(benzo[d][1,3]dioxol-5-yl)-4-oxobutanoic
acid; [0078]
2-(benzo[d][1,3]dioxol-5-yl)-4-(2-methylbenzo[d][1,3]dioxol-5-yl)-4-oxobu-
tanoic acid; [0079]
4-(benzo[d][1,3]dioxol-5-yl)-3-methyl-4-oxo-2-(3,4,5-trimethoxybenzyl)but-
anoic acid; [0080] and the physiologically acceptable salts and/or
prodrugs thereof.
[0081] Particularly preferred representative compounds according to
general formulas (A), (A-1) and (A-2), respectively, are:
##STR00003##
[0082] A second aspect of the invention relates to a compound
according to general formula (B)
##STR00004##
wherein [0083] R.sub.B1 means --H, --F, --Cl, --Br, --I, --CN,
--NO.sub.2, --CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X,
--C(.dbd.O)H, --C(.dbd.O)OH, --C(.dbd.O)OR.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.().sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0084] R.sub.B2 and
R.sub.B3, respectively independently, mean --F, --Cl, --Br, --I,
--CN, --CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H,
--C(.dbd.O)OH, --C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, --C(.dbd.O)N(R.sub.X).sub.2, --OH,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2-R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0085] X.sub.B1 and
X.sub.B2, respectively independently, mean O, S or NH; [0086]
m.sub.B means 1, 2, 3, 4, 5, or 6; and [0087] n.sub.B means 1, 2,
3, 4, 5, or 6; [0088] or a physiologically acceptable salt and/or a
prodrug thereof.
[0089] In a preferred embodiment of the compound according to
general formula (B), [0090] R.sub.B1 means --H or
--C.sub.1-8-aliphatic; [0091] R.sub.B2 and R.sub.B3, respectively
independently, mean --C(.dbd.O)R.sub.X, --C(.dbd.O)H,
--C(.dbd.O)OH, --C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, or --C(.dbd.O)N(R.sub.X).sub.2; [0092]
X.sub.B1 and X.sub.B2, respectively independently, mean O or S;
[0093] m.sub.B means 1, 2, 3, or 4; and [0094] n.sub.B means 1, 2,
3, or 4.
[0095] In another preferred embodiment according to general formula
(B), [0096] R.sub.B1 means --H or --C.sub.1-8-alkyl; [0097]
R.sub.B2 and R.sub.B3, respectively independently, mean
--C(.dbd.O)OH or --C(.dbd.O)OR.sub.X; [0098] X.sub.B1 and X.sub.B2,
respectively independently, mean O or S; [0099] m.sub.B means 1 or
2; and [0100] n.sub.B means 1 or 2.
[0101] In a further preferred embodiment according to general
formula (B), [0102] R.sub.B1 means --H or --CH.sub.3; [0103]
R.sub.B2 and respectively independently, mean --C(.dbd.O)OH; [0104]
X.sub.B1 and X.sub.B2, respectively independently, mean O; [0105]
m.sub.B means 1; and [0106] n.sub.B means 1.
[0107] Particularly preferred embodiments of the compound according
to general formula (B) have the general formulas (B-1) or
(B-2):
##STR00005##
[0108] Compounds of the general formulas (B-1) or (B-2) that are
particularly preferred are those, wherein [0109] R.sub.B1 means --H
or --CH.sub.3; [0110] m.sub.B means 1; and [0111] n.sub.8 means
1.
[0112] Compounds from the following group are most particularly
preferred: [0113] 2-[2-(carboxymethyloxy)-4-methyl-phenoxy]acetic
acid, [0114] 2,2'-(1,3-phenylenebis(oxy))diacetic acid; [0115] and
the physiologically acceptable salts and/or prodrugs thereof.
[0116] Particularly preferred representative compounds according to
general formulas (B), (B-1) and (B-2), respectively, are:
##STR00006##
[0117] A third aspect of the invention relates to a compound
according to general formula (C)
##STR00007##
wherein [0118] R.sub.C1 means O, S, or NR.sub.C3; [0119] R.sub.C1
means --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NHR.sub.X, --N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0120] R.sub.C2 means
--H or --(CH.sub.2).sub.1-6--X.sub.C--R.sub.X; [0121] R.sub.C3
means --R.sub.X, [0122] R.sub.C4 means --F, --Cl, --Br, --I, --CN,
--NO.sub.2, --CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X,
--C(.dbd.O)H, --C(.dbd.O)OH, --C(.dbd.O)OR.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0123] X.sub.C means
O, S, or NH; and [0124] m.sub.C means 0, 1, 2, 3, 4, 5 or 6; [0125]
or a physiologically acceptable salt and/or a prodrug thereof.
[0126] In a preferred embodiment of the compound according to
general formula (C), [0127] A.sub.C means O or NR.sub.C3; [0128]
R.sub.C1 means --R.sub.X, [0129] R.sub.C2 means --H or
--CH.sub.2--X.sub.C--CH.sub.2-heteroaryl, [0130] R.sub.C3 means
--(CH.sub.2).sub.1-8--C(.dbd.O)OH; [0131] R.sub.C4 means
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X, or
--C(.dbd.O)N(R.sub.X).sub.2; [0132] X.sub.C means O or S; and
[0133] m.sub.C means 0, 1, 2, 3, or 4.
[0134] In another preferred embodiment according to general formula
(C), [0135] A.sub.C means O or NR.sub.C3; [0136] R.sub.C1 means
--C.sub.1-8-aliphatic or -aryl; [0137] R.sub.C2 means --H, or
--CH.sub.2--O--CH.sub.2-heteroaryl or
--CH.sub.2--S--CH.sub.2-heteroaryl; [0138] R.sub.C3 means
--(CH.sub.2).sub.1-6--C(.dbd.O)OH; [0139] R.sub.C4 means
--C(.dbd.O)OH or --C(.dbd.O)OR.sub.X; [0140] X.sub.C means O or S;
and [0141] m.sub.C means 0, 1, or 2.
[0142] In a further preferred embodiment according to general
formula (C), [0143] A.sub.C means O or NR.sub.C3; [0144] R.sub.C1
means --C.sub.1-8-alkyl or -phenyl, optionally substituted; [0145]
R.sub.C2 means --H or --CH.sub.2--S--CH.sub.2-heteroaryl; [0146]
R.sub.C3 means --(CH.sub.2).sub.2--C(.dbd.O)OH; [0147] R.sub.C4
means --C(.dbd.O)OH; [0148] X.sub.C means S, and [0149] m.sub.C
means 0 or 2.
[0150] Particularly preferred embodiments of the compound according
to general formula (C) have the general formula (C-1):
##STR00008##
[0151] Compounds of the general formula (C-1) that are particularly
preferred are those, wherein [0152] A.sub.C means O or NR.sub.C3;
[0153] R.sub.C1 means CH.sub.3 or -phenyl, optionally substituted;
[0154] R.sub.C2 means --H or --CH.sub.2--S--CH.sub.2-heteroaryl,
optionally substituted; [0155] R.sub.C3 means
--(CH.sub.2).sub.2--C(.dbd.)OH; and [0156] m.sub.C means 0 or
2.
[0157] Compounds of the general formula (C-1) that are more
particularly preferred are those, wherein [0158] A.sub.C means O or
NR.sub.C3; [0159] R.sub.C1 means CH.sub.3 or 4-methoxyphenyl;
[0160] R.sub.C2 means --H or
--CH.sub.2--S--CH.sub.2--(2-carboxy-5-methylfuran-4-yl); [0161]
R.sub.C3 means --(CH.sub.2).sub.2--C(.dbd.O)OH; and [0162] m.sub.C
means 0 or 2.
[0163] Compounds from the following group are most particularly
preferred: [0164]
3,3'-(5-(4-methoxyphenyl)-1H-pyrrole-1,2-diyl)dipropanoic acid;
[0165]
5,5-dimethyl-4,4'-(sulfanediylbis(methyl))-di(furan-2-carboxylic
acid); [0166] and the physiologically acceptable salts and/or
prodrugs thereof.
[0167] Particularly preferred representative compounds according to
general formulas (C) and (C-1), respectively, are:
##STR00009##
[0168] A fourth aspect of the invention relates to a compound
according to general formula (D)
##STR00010##
wherein [0169] X.sub.D1 means O, S, or NR.sub.D3; [0170] X.sub.D2
means O or S; [0171] Y.sub.D means --(CH.sub.2).sub.0-6-- or --C(50
O)--(CH.sub.2).sub.1-6--X.sub.D2--; [0172] A.sub.D1 means N, NH or
CH; [0173] A.sub.D2 means N or C; [0174] A.sub.D3 means N or CH;
[0175] R.sub.D1 means --H, --F, --Cl, --Br, --I, --CN, --NO.sub.2,
--CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H,
--C(.dbd.O)OH, --C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, --C(.dbd.O)N(R.sub.X).sub.2, --OH,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-;
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0176] R.sub.D2 means
--H, --F, --Cl, --Br, --I, --ON, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH, --C(.dbd.O)OR.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--O(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SO.sub.3H,
--S(.dbd.O).sub.1-2-R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2, --N.sup.30
(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X) or --PO(OR.sub.X).sub.2; [0177] R.sub.D3 means --H,
--F, --Cl, --Br, --ON, --NO.sub.2, --CHO, --C(.dbd.O)R.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)N(R.sub.X).sub.2, --SH,
--SR.sub.X, --NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.2O.sup.-, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0178] R.sub.D4 means
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X, or
--C(.dbd.O)N(R.sub.X).sub.2; [0179] or a physiologically acceptable
salt and/or a prodrug thereof.
[0180] In a preferred embodiment of the compound according to
general formula (D), [0181] X.sub.D1 means O or NR.sub.D3; [0182]
X.sub.C2 means O or S; [0183] Y.sub.D means --(CH.sub.2).sub.1-4-
or --C(.dbd.O)--(CH.sub.2).sub.1-4--X.sub.D2--; [0184] A.sub.D1
means N, NH or CH; [0185] A.sub.D2 means N or C; [0186] A.sub.D3
means N or CH; [0187] with the proviso that 2 of A.sub.D1, A.sub.D2
and A.sub.D3 mean N and NH, respectively, while the other means C
and CH, respectively; [0188] R.sub.C1 means --H, --F, --Cl, --Br,
--I, --CN, or --NO.sub.2; [0189] R.sub.D2 means --H or --R.sub.X;
[0190] R.sub.D3 means --H or --R.sub.X; and [0191] R.sub.D4 means
--C(.dbd.O)OH or --C(.dbd.O)OR.sub.X;
[0192] In another preferred embodiment according to general formula
(D), [0193] X.sub.D1 means O or NR.sub.D3; [0194] X.sub.D2 means S;
[0195] Y.sub.D means --CH.sub.2-- or --C(.dbd.O)--(CH.sub.2)--S--;
[0196] A.sub.D1 means N or CH; [0197] A.sub.D2 means N or C; [0198]
A.sub.D3 means N or CH; [0199] with the proviso that 2 of D.sub.D1,
D.sub.D2 and D.sub.D3 mean N and NH, respectively, while the other
means C and CH, respectively; [0200] R.sub.D1 means --H or --Cl;
[0201] R.sub.D2 means --H; [0202] R.sub.D3 means --H; and [0203]
R.sub.D4 means --CO.sub.2H.
[0204] Particularly preferred embodiments of the compound according
to general formula (D) have the general formula (D-1):
##STR00011##
[0205] Compounds of the general formula (D-1) that are particularly
preferred are those, wherein [0206] X.sub.D1 means O or NR.sub.D3;
[0207] Y.sub.D means --S--(CH.sub.2)--C(.dbd.O)-- or
--C(.dbd.O)--(CH.sub.2)--S--; [0208] A.sub.D1 means NH or CH;
[0209] A.sub.D2 means N or C, [0210] with the proviso that A.sub.D1
means CH or NH, while A.sub.D2 means N or C, respectively; [0211]
R.sub.D1 means --H or --Cl; [0212] R.sub.D2 means --H; [0213]
R.sub.D3 means --H.
[0214] Compounds from the following group are most particularly
preferred: [0215] 2-(imidazo[1,2-a]pyridin-2-ylmethoxy)benzoic
acid; [0216]
5-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)-2-chlorobenzoic acid;
[0217] and the physiologically acceptable salts and/or prodrugs
thereof.
[0218] Particularly preferred representative compounds according to
general formulas (D) and (D-1), respectively, are:
##STR00012##
[0219] A fifth aspect of the invention relates to a compound
according to general formula (E)
##STR00013##
wherein [0220] R.sub.E1 and R.sub.E2, respectively independently,
mean --H, --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0221] R.sub.E3 and
R.sub.E4, respectively independently, mean --H, --F, --Cl, --Br,
--I, --CN, --NO.sub.2, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --O(OR.sub.X).sub.2; [0222] R.sub.E5 means
--F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0223] m.sub.E means
0, 1, 2, 3, 4, 5, or 6; and [0224] n.sub.E means 0, 1, 2, 3, 4, 5,
or 6; [0225] or a physiologically acceptable salt and/or a prodrug
thereof.
[0226] In a preferred embodiment of the compound according to
general formula (E), [0227] R.sub.E1 and R.sub.E2, respectively
independently, mean --OC.sub.1-8-aliphatic [0228] R.sub.E3 and
R.sub.E4, respectively independently, mean --H or --R.sub.X; [0229]
R.sub.E5 means --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X, or
C(.dbd.O)N(R.sub.X).sub.2; [0230] m.sub.E means 0, 1, 2, 3, or 4;
and [0231] n.sub.E means 0,1, 2, 3, or 4
[0232] In another preferred embodiment according to general formula
(E), [0233] R.sub.E1 and R.sub.E2, respectively independently, mean
--OC.sub.1-8-alkyl; [0234] R.sub.E3 and R.sub.E4, respectively
independently, mean --H or --R.sub.X; [0235] R.sub.E5 means
--C(.dbd.O)OH or --C(.dbd.O)OR.sub.X; [0236] m.sub.E means 0, 1, or
2; and [0237] n.sub.E means 0, 1, or 2.
[0238] In a further preferred embodiment according to general
formula (E), [0239] R.sub.E1 and R.sub.E2, respectively
independently, mean --OCH.sub.3; [0240] R.sub.E3 and R.sub.E4,
respectively independently, mean --H; [0241] R.sub.E5 means
--C(.dbd.O)OH; [0242] m.sub.E means 1, and [0243] n.sub.E means
1.
[0244] Particularly preferred embodiments of the compound according
to general formula (E) have the general formula (E-1):
##STR00014##
[0245] Compounds of the general formula (E-1) that are particularly
preferred are those, wherein [0246] R.sub.E1 and R.sub.E2,
respectively independently, mean --OCH.sub.3; [0247] R.sub.E3 and
R.sub.E4, respectively independently, mean --H; [0248] m.sub.E
means 1; and [0249] n.sub.E means 1.
[0250] Compounds from the following group are most particularly
preferred: [0251]
2-(1-((4,7-dimethoxy-1H-indole-2-carboxamido)methyl)cyclohexyl)a-
cetic acid; [0252] and the physiologically acceptable salts and/or
prodrugs thereof.
[0253] A particularly preferred representative compound according
to general formulas (E) and (E-1), respectively, is:
##STR00015##
[0254] A sixth aspect of the invention relates to a compound
according to general formula (F)
##STR00016##
wherein [0255] R.sub.F1 means --F, --Cl, --Br, --I, --ON, --CHO,
.dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--O(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X,
--S(.dbd.O).sub.1-2-R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.2 or --PO(OR.sub.X).sub.2; [0256] R.sub.F2 means
--H, --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.H,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R).sub.3 or --PO(OR.sub.X).sub.2; [0257] R.sub.F3 means --H,
--F, --Cl, --Br, --ON, --NO.sub.2, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--O(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --S(.dbd.O).sub.1-2--R.sub.X,
--S(.dbd.O).sub.1-2NH.sub.2, --NH.sub.2, --NHR.sub.X,
--N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2;
[0258] R.sub.F4 means --H, --F, --Cl, -Br, --I, --CN, --NO.sub.2,
--CHO, .dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H,
--O(.dbd.O)OH, --C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, --C(.dbd.O)N(R.sub.X).sub.2, --OH,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0259] R.sub.F5,
R.sub.F6 and R.sub.F7, respectively independently, mean --H, --F,
--Cl, --Br, --I, --CN, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--O(.dbd.O)OR.sub.X, --O(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHO(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0260] X.sub.F means
O, S, or NR.sub.F5; [0261] Y.sub.F means
--(CH.sub.2).sub.1-6C(.dbd.O)NH--(CH.sub.2).sub.1-6-; [0262]
A.sub.F1 means O, S, or NR.sub.F6; [0263] A.sub.F2 means O, S, or
NR.sub.F7; [0264] or a physiologically acceptable salt and/or a
prodrug thereof.
[0265] In a preferred embodiment of the compound according to
general formula (F), [0266] R.sub.F1 means --C.sub.1-8-aliphatic;
[0267] R.sub.F2 means --C.sub.1-8-aliphatic; [0268] R.sub.F3 means
--C.sub.1-8-aliphatic; [0269] R.sub.F4 means --C(.dbd.O)R.sub.X,
--O(.dbd.O)H, --C(.dbd.O)OH, --C(.dbd.O)OR.sub.X,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X, or
--C(.dbd.O)N(R.sub.X).sub.2; [0270] R.sub.F5, R.sub.F6 and
R.sub.F7, respectively independently, mean --H, [0271] X.sub.F
means O; [0272] Y.sub.F means
--(CH.sub.2).sub.1-6C(.dbd.O)NH--(CH.sub.2).sub.1-6-; [0273]
A.sub.F1 means O; and [0274] A.sub.F2 means O.
[0275] In another preferred embodiment according to general formula
(F), [0276] R.sub.F1 means --C.sub.1-8-alkyl; [0277] R.sub.F2 means
--C.sub.1-8-alkyl; [0278] R.sub.F3 means --C.sub.1-8-alkyl; [0279]
R.sub.F4 means --C(.dbd.O)OH or --C(.dbd.O)OR.sub.X; [0280]
R.sub.F5, R.sub.F6 and R.sub.F7, respectively independently, mean
--H; [0281] X.sub.F means O; [0282] Y.sub.F means
--(CH.sub.2).sub.1-4C(.dbd.O)NH--(CH.sub.2).sub.1-4-; [0283]
A.sub.F1 means O; and [0284] A.sub.F2 means O.
[0285] In a further preferred embodiment according to general
formula (F), [0286] R.sub.F1 means --CH.sub.3; [0287] R.sub.F2
means --CH.sub.3; [0288] R.sub.F3 means --C(CH.sub.3).sub.3; [0289]
R.sub.F4 means --CO.sub.2H; [0290] R.sub.F5, R.sub.F6 and R.sub.F7,
respectively independently, mean --H; [0291] X.sub.F means O;
[0292] Y.sub.F means --CH.sub.2C(.dbd.O)NH--(CH.sub.2).sub.2--;
[0293] A.sub.F1 means O; and [0294] A.sub.F2 means O.
[0295] Particularly preferred embodiments of the compound according
to general formula (F) have the general formula (F-1):
##STR00017##
[0296] Compounds of the general formula (F-1) that are particularly
preferred are those, wherein [0297] R.sub.F1 means --CH.sub.3;
[0298] R.sub.F2 means --CH.sub.3; [0299] R.sub.F3 means
--C(CH.sub.3).sub.3; and [0300] Y.sub.F means
--CH.sub.2C(.dbd.O)NH--(CH.sub.2).sub.2--;
[0301] Compounds from the following group are most particularly
preferred: [0302]
3-(2-(3-tert-butyl-5,9-dimethyl-7-oxo-7H-furo[3,2-g]chromen-6-yl-
)acetamido)propanoic acid; [0303] and the physiologically
acceptable salts and/or prodrugs thereof.
[0304] A particularly preferred representative compound according
to general formulas (F) and (F-1), respectively, is:
##STR00018##
[0305] For the purpose of the specification, RUNX1/ETO is the
fusion protein containing the DNA-binding domain (Runt, RHD) of the
RUNX1 transcription factor but lacking the C-terminal
transactivation sequence being replaced by almost the entire ETO
protein. A compound inhibiting RUNX1/ETO-tetramerization is a
compound that reduces the tendency of RUNX1/ETO to form tetramers.
For the purpose of the specification, NHR2 is a functional domain
of RUNX1/ETO, namely the nervy homology region 2. A compound
inhibiting NHR2 is a compound that [0306] a) reduces the tendency
of NHR2 to form tetramers, and/or [0307] b) reduces the tendency of
NHR2-containing proteins that include, but are not limited to, wild
type ETO proteins, homologs of ETO proteins and CBFA2T3-GLIS2
fusion proteins (Gruber et al Cancer Cell, 2012, 22, 683-697; C.
Thiollier et al., The Journal of Experimental Medicine 2012,
209(11), 2017-2031) to form tetramers, and/or [0308] c) interferes
with the onset and/or maintenance of AML and other NHR2 dependent
leukemias, and/or [0309] d) interferes with the role of
NHR2-containing proteins in other disorders or diseases that
include, but are not limited to, cancer or a hematopoietic disease,
preferably leukemia, particularly preferably selected from the
group consisting of myeloid leukemia, acute myeloid leukemia,
promyeloid leukemia, acute promyeloid leukemia, promyelocytic
leukemia, acute promyelocytic leukemia, megakaryoblastic leukemia
and acute megakaryoblastic leukemia, and/or [0310] e) enables a
personalized therapy of NHR2-dependent disorders or diseases,
and/or [0311] f) interferes with blocking of myeloid
differentiation caused by NHR2 tetramerization, and/or [0312] g)
interferes with the proliferation of RUNX1/ETO-dependent cells that
include, but are not limited to, SKNO-1 cells, and/or [0313] h)
interferes with the enhanced self-renewal capacity of hematopoietic
progenitors, and/or [0314] i) interferes with the induction or
maintenance of disorders or diseases that include, but are not
limited to, cancer or a hematopoietic disease, preferably leukemia,
particularly preferably selected from the group consisting of
myeloid leukemia, acute myeloid leukemia, promyeloid leukemia,
acute promyeloid leukemia, promyelocytic leukemia, acute
promyelocytic leukemia, megakaryoblastic leukemia and acute
megakaryoblastic leukemia in mammals (Y. Liu et at., Cancer Cell,
2006, 9, 249-260: C. Wichmann et al. Blood, 2010, 116(4), 602-613;
and C. Wichmann et al., Cancer Research, 2007, 67, 2280-2289).
[0315] For further details, reference is made to e.g. D. G. Tenen,
Nat Rev Cancer, 2003; 3(2): 89-101; J. Zhang et al., Science, 2004;
305(5688): 1286-1289; Metz et al., Current Pharmaceutical Design,
2012, 18, 4630-4647.
[0316] For the purpose of the specification, in the definitions of
the compounds according to general formulas (A), (B), (C), (D), (E)
and (F), [0317] in each case R.sub.X, respectively independently,
means --C.sub.1-8-aliphatic, --C.sub.3-12-cycloaliphatic, -aryl,
heteroaryl, --C.sub.1-8-aliphatic-C.sub.3-12-cycloaliphatic,
--C.sub.1-8-aliphatic-aryl, --.sub.1-8-aliphatic-heteroaryl,
--C.sub.3-8-cycloaliphatic-C.sub.1-8-aliphatic,
--C.sub.3-8-cycloaliphatic-aryl or
--C.sub.3-8-cycloaliphatic-heteroaryl; [0318] in each case
"aliphatic", respectively independently, means a branched or
unbranched, saturated or a mono- or polyunsaturated, unsubstituted
or mono- or polysubstituted, aliphatic hydrocarbon residue; [0319]
in each case "cycloaliphatic", respectively independently, means a
saturated or a mono- or polyunsaturated, unsubstituted or mono- or
polysubstituted, alicyclic, mono- or multicyclic hydrocarbon
residue; [0320] in each case with respect to "aliphatic" and
"cycloaliphatic", "mono- or polysubstituted", respectively
independently, means the mono- or polysubstitution of one or more
hydrogen atoms by --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO,
.dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2; [0321] in each case
"aryl", respectively independently, means a carbacyclic ring system
with at least one aromatic ring, but without heteroatoms in this
ring, wherein, if necessary, the aryl residues can be condensed
with further saturated, (partially) unsaturated or aromatic ring
systems, and each aryl residue can be present in unsubstituted or
mono- or polysubstituted form, wherein the aryl substituents can be
the same or different and in any desired and possible position of
the aryl; [0322] in each case "heteroaryl", respectively
independently, means a 5-, 6- or 7-membered cyclic aromatic
residue, which contains 1, 2, 3, 4 or 5 heteroatoms, wherein the
heteroatoms, the same or different, are nitrogen, oxygen or
sulphur, and the heterocycle can be unsubstituted or mono- or
polysubstituted; wherein in the case of the substitution on the
heterocycle the substituents can be the same or different and can
be in any desired and possible position of the heteroaryl; and
wherein the heterocycle can also be part of a bi- or polycyclic
system; [0323] in each case with respect to "aryl", and
"heteroaryl", "mono- or polysubstituted", respectively
independently, means the mono- or polysubstitution of one or more
hydrogen atoms of the ring system by substituents selected from the
group comprising --F, --Cl, --Br, --I, --CN, --NO.sub.2, --CHO,
.dbd.O, --R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)--N(R.sub.X).sub.2, --OH, --O(CH.sub.2).sub.1-2O--,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X,
--NH--C(.dbd.O)NH.sub.2, --NHC(.dbd.O)NHR.sub.X,
--NHC(.dbd.O)--N(R.sub.X).sub.2, --Si(R.sub.X).sub.3 and
--PO(OR.sub.X).sub.2; wherein if necessary N-ring atoms present can
be respectively oxidized; [0324] in the form of a single
stereoisomer or mixture thereof, the free compounds and/or their
physiologically compatible salts and/or prodrugs and/or
solvates.
[0325] In the combination of different residues, e.g. R.sub.A3,
R.sub.A4, and R.sub.A5, and also the combination of residues at
substituents thereof such as e.g. --OR.sub.X, --SR.sub.X,
--S(.dbd.O).sub.1-2R.sub.X or --C(.dbd.O)OR.sub.X, a substituent,
e.g. R.sub.X, can assume different meanings within a substance for
two or more residues, e.g. R.sub.A3, R.sub.A4, and R.sub.A5.
[0326] For the purposes of the description hydrocarbon residues are
divided into aliphatic hydrocarbon residues and aromatic
hydrocarbon residues
[0327] Aliphatic hydrocarbon residues are themselves divided into
non-cyclic aliphatic hydrocarbon residues (="aliphatic") and cyclic
aliphatic hydrocarbon residues, i.e. alicyclic hydrocarbon residues
(="cycloaliphatic"). Cycloaliphatic compounds can be monocyclic or
multicyclic Alicyclic hydrocarbon residues ("cycloaliphatic")
comprise both pure aliphatic carbocycles and aliphatic
heterocycles, i.e.--unless expressly specified--"cycloaliphatic"
comprises pure aliphatic carbocycles (e.g. cyclohexyl), pure
aliphatic heterocycles (e.g. piperidyl or piperazyl) and also
non-aromatic, multicyclic, possibly mixed, systems (e.g. decalinyl,
decahydro-quinolinyl).
[0328] Aromatic hydrocarbons are themselves divided into
carbocyclic aromatic hydrocarbons (="aryl") and heterocyclic
aromatic hydrocarbons (="heteroaryl").
[0329] The classification of multicyclic, at least partially
aromatic systems preferably depends on whether at least one
aromatic ring of the multicyclic system has at least one heteroatom
(usually N, O or S) in the ring. If at least one such heteroatom is
present in this ring, this is preferably a "heteroaryl" (even if a
further carbocyclic aromatic or non-aromatic ring with or without
heteroatom is possibly present as additionally present cycle of the
multicyclic system); if such a heteroatom is not present in any of
the possibly several aromatic rings of the multicyclic system, then
this is preferably "aryl" (even if a ring heteroatom is present in
a possibly additionally present non-aromatic cycle of the
multicyclic system).
[0330] Therefore, the following priority in the classification
applies within the cyclic substituents:
heteroaryl>aryl>cycloaliphatic.
[0331] For the purposes of the description monovalent and
multivalent, i.e. bivalent, hydrocarbon residues are not
distinguished between conceptually, i.e, depending on the context,
"C.sub.1-8-aliphatic" covers e.g. --C.sub.1-8-alkyl,
--C.sub.1-8-alkenyl and --C.sub.1-8-alkinyl, as well as e.g.
--C.sub.1-8-alkylene-, --C.sub.1-8-alkenylene- and
C.sub.1-8-alkinylene.
[0332] Aliphatic means preferably respectively a branched or
unbranched, saturated or a mono- or polyunsaturated, unsubstituted
or mono- or polysubstituted, aliphatic hydrocarbon residue.
[0333] Where aliphatic is mono- or polysubstituted, the
substituents are selected independently of one another from the
group comprising --F, --Cl, --Br, --CN, --NO.sub.2, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 or --PO(OR.sub.X).sub.2.
[0334] Thus, "aliphatic" covers acyclic saturated or unsaturated
hydrocarbon residues that can be branched or straight-chain, i.e.
alkenyls, alkenyls and alkinyls. In this case, alkenyls have at
least one C.dbd.C double bond and alkinyls have at least one
C.ident.C triple bond. Preferred unsubstituted monovalent
aliphatics comprise --CH.sub.3, --CH.sub.2CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.3, --CH(CH.sub.3).sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3, --CH(CH.sub.3)CH.sub.2CH.sub.3,
--CH.sub.2CH(CH.sub.3).sub.2, --C(CH.sub.3).sub.3,
--CH.sub.2CH.sub.2CH.sub.2--CH.sub.2CH.sub.3 and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3; but also
--CH.dbd.CH.sub.2, --CH.sub.2CH.dbd.CH.sub.2, --CH.dbd.CHCH.sub.3,
--CH.sub.2C.ident.CH, --C.ident.CCH.sub.3 and
--CH.dbd.CHCH.dbd.CH.sub.2. Preferred unsubstituted bivalent
aliphatics comprise --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)--, --CH(CH.sub.3)--CH.sub.2--,
--CH(CH.sub.2CH.sub.3)CH.sub.2--, --CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)--CH.sub.2--,
--CH.sub.2CH.sub.2CH(CH.sub.3)--,
--CH--(CH.sub.2CH.sub.3)CH.sub.2-- and
--CH.sub.2CH.sub.2--CH.sub.2CH.sub.2--; but also --CH.dbd.CH--,
--CH.ident.CH--, --CH.sub.2CH.dbd.CH--, --CH.dbd.CHCH.sub.2--,
--CH.sub.2C.ident.C-- and --C.ident.CCH.sub.2--. Preferred
substituted monovalent aliphatics comprise --CH.sub.2F,
--CHF.sub.2, --CF.sub.3, --CH.sub.2CF.sub.3, --CF.sub.2CF.sub.3,
--CH.sub.2OH, --CH.sub.2CH.sub.2OH, --CH.sub.2CHOHCH.sub.3,
--CH.sub.2OCH.sub.3, --CH.sub.2CH.sub.2OCH.sub.3 and
--CH.sub.2N(CH.sub.3).sub.2. Preferred substituted bivalent
aliphatics comprise --CF.sub.2--, --CF.sub.2CF.sub.2--,
--CH.sub.2CHOH--, --CHOHCH.sub.2-- and --CH.sub.2CHOHCH.sub.2--,
-Methyl-, -ethyl-, -n-propyl- and -n-butyl- are particularly
preferred.
[0335] Cycloaliphatic means preferably respectively a saturated or
a mono- or polyunsaturated, unsubstituted or mono- or
polysubstituted. aliphatic (i.e. not aromatic), mono- or
multicyclic hydrocarbon residue. The number of ring-carbon atoms
preferably lies in the specified range (i.e. a
"C.sub.3-12-cycloaliphatic" preferably has 3, 4, 5, 6, 7, 8, 9, 10,
11 or 12 ring-carbon atoms). For the purposes of the description
"C.sub.3-12-cycloaliphatic" is preferably a cyclic hydrocarbon with
3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 ring-carbon atoms, saturated or
unsaturated, but not aromatic, wherein possibly one or two carbon
atoms are replaced independently of one another by a heteroatom S,
N or O. Where cycloalkyl is mono- or polysubstituted, the
substituents are selected independently of one another from the
group comprising --F, --Cl, --Br, --I, --CN, --CHO, .dbd.O,
--R.sub.X, --C(.dbd.O)R.sub.X, --O(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.)OR.sub.X; --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)N(R.sub.X).sub.2, --OH, --OR.sub.X, --OC(.dbd.O)H,
--OC(.dbd.O)R.sub.X, --OC(.dbd.O)--OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2NH.sub.2, --NHR.sub.X, --N(R.sub.X).sub.2,
--N.sup.+(R.sub.X).sub.3, --N.sup.+(R.sub.X).sub.2O.sup.-,
--NHC(.dbd.O)R.sub.X, --NHC(.dbd.O)OR.sub.X, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X. --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3, or --PO(OR.sub.X).sub.2.
[0336] Preferably, C.sub.3-12-cycloaliphatic is selected from the
group comprising cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cycloactyl, cyclononyl, cyclodecyl, cycloundecyl,
cyclododecyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,
cyclooctenyl, cyclononenyl, cyclodecenyl, cycloundecenyl,
cyclododecenyl, but also tetrahydropyranyl, dioxanyl, dioxolanyl,
morpholinyl, piperidinyl, piperazinyl, pyrazolinonyl and
pyrrolidinyl.
[0337] In association with "aliphatic" or "cycloaliphatic", "mono-
or polysubstituted" is preferably understood to mean the mono- or
polysubstitution, e.g. the mono-, di-, tri- or 4-substitution, of
one or more hydrogen atoms by --F, --Cl, --Br, --I, --OH,
--OC.sub.1-8-alkyl, --OC(.dbd.O)C.sub.1-8-alkyl, --SH, --NH.sub.2,
--NHC.sub.1-8-alkyl, --N(C.sub.1-8-alkyl).sub.2,
--C(.dbd.O)OC.sub.1-8-alkyl or --C(.dbd.O)OH. Particularly
preferred substituents are --F, --Cl, --OH, --SH, --NH.sub.2 and
--C(.dbd.O)OH.
[0338] Polysubstituted residues are understood to be those residues
that are polysubstituted, e.g. twice or three times either at
different or at the same atoms, e.g. three times at the same
C--atom, as in the case of --CF.sub.3 or --CH.sub.2CF.sub.3, or at
different sites, as in the case of --CH(OH)--CH.dbd.CH--CHCl.sub.2.
The polysubstitution can occur with the same or with different
substituents. A substituent may also be substituted itself. Thus,
--Oaliphatic also covers --OCH.sub.2CH.sub.2O--CH.sub.2CH.sub.2OH,
amongst others. It is preferred if aliphatic or cycloaliphatic is
substituted with --F, --Cl, --Br, --CN, --CH.sub.3,
--C.sub.2H.sub.5, --NH.sub.2, --NO.sub.2, --SH, --CF.sub.3, --OH,
--OCH.sub.3, --OC.sub.2H.sub.5 or --N(CH.sub.3).sub.2. It is most
particularly preferred if aliphatic or cycloaliphatic is
substituted with --OH, --OCH.sub.3 or --OC.sub.2H.sub.5.
[0339] Aryl preferably respectively independently stands for a
carbocyclic ring system with at least one aromatic ring, but
without heteroatoms in this ring, wherein the aryl residues can
possibly be condensed with further saturated, (partially)
unsaturated or aromatic ring systems and each aryl residue can be
present in unsubstituted or mono- or polysubstituted form, wherein
the aryl substituents are the same or different and can be in any
desired and possible position of the aryl. Preferred aryls are
phenyl, naphthyl, anthracenyl, phenanthrenyl, fiuoroanthenyl,
fluoroenyl, indanyl and tetralinyl. Phenyl and naphthyl are
particularly preferred. Where aryl is mono- or polysubstituted, the
aryl substituents can be the same or different and be in any
desired and possible position of the aryl, and are selected
independently of one another from the group comprising --F, --Cl,
--Br, --CN, --NO.sub.3, --CHO, .dbd.O, --R.sub.X,
--C(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)--N(R.sub.X).sub.2, --OH, --O(CH.sub.2).sub.1-2O--,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SR, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--NHR.sub.X, --N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NH--C(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 and --PO(OR.sub.X).sub.2; wherein if necessary
N-ring atoms present can be respectively oxidized. Preferred
substituted aryls are 2-fluorophenyl, 3-fluorophenyl,
4-fluorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl,
3,4-difiuorophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl,
2,3-dichlorophenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl,
2-methoxy-phenyl, 3-methoxy-phenyl, 4-methoxy-phenyl,
2,3-dimethoxy-phenyl, 2,4-dimethoxy-phenyl, 3,4-dimethoxy-phenyl,
2-methyl-phenyl, 3-methyl-phenyl, 4-methyl-phenyl,
2,3-dimethyl-phenyl, 2,4-dimethyl-phenyl and
3,4-dimethyl-phenyl.
[0340] Heteroaryl preferably stands for a 5-, 6- or 7-membered
cyclic aromatic residue that contains 1, 2, 3, 4 or 5 heteroatoms,
wherein the heteroatoms, the same or different, are nitrogen,
oxygen or sulphur, and the heterocycle can be unsubstituted or
mono- or polysubstituted; wherein in the case of the substitution
on the heterocycle, the substituents can be the same or different
and can be in any desired and possible position of the heteroaryl;
and wherein the heterocycle can also be part of a bi- or polycyclic
system. "Heteroaryl" is preferably selected from the group
comprising pyrrolyl, indolyl, furyl (furanyl), benzofuranyl,
thienyl (thiophenyl), benzothienyl, benzothiadiazolyl,
benzooxadiazolyl, benzothiazolyl, benzooxazolyl, benzotriazolyl,
benzodioxolanyl, benzodioxanyl, phthalazinyl, pyrazolyl,
imidazolyl, thiazolyl, oxazolyl, isoxazoyl, pyridinyl, pyridazinyl,
pyrimidinyl, pyrazinyl, pyranyl, indazolyl, purinyl, indolizinyl,
quinolinyl, isoquinolinyl, quinazolinyl, carbazolyl, phenazinyl,
phenothiazinyl or oxadiazolyl, wherein the bonding can occur via
any desirable and possible ring member of the heteroaryl residue.
Where heteroaryl is mono- or polysubstituted, the heteroaryl
substituents can be the same or different and can be in any
desirable and possible position of the heteroaryl, and are selected
independently of one another from the group comprising --F, --Cl,
--Br, --I, --CN, --NO.sub.2, --CHO, .dbd.O, --R.sub.X,
--O(.dbd.O)R.sub.X, --C(.dbd.O)H, --C(.dbd.O)OH,
--C(.dbd.O)OR.sub.X, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHR.sub.X,
--C(.dbd.O)--N(R.sub.X).sub.2, --OH, --O(CH.sub.2).sub.1-2O--,
--OR.sub.X, --OC(.dbd.O)H, --OC(.dbd.O)R.sub.X,
--OC(.dbd.O)OR.sub.X, --OC(.dbd.O)NHR.sub.X,
--OC(.dbd.O)N(R.sub.X).sub.2, --SH, --SR.sub.X, --SO.sub.3H,
--S(.dbd.O).sub.1-2--R.sub.X, --S(.dbd.O).sub.1-2NH.sub.2,
--N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3,
--N.sup.+(R.sub.X).sub.2O.sup.-, --NHC(.dbd.O)R.sub.X,
--NHC(.dbd.O)OR.sub.X, --NH--C(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHR.sub.X, --NHC(.dbd.O)--N(R.sub.X).sub.2,
--Si(R.sub.X).sub.3 and --PO(OR.sub.X).sub.2; wherein if necessary
N-ring atoms present can be respectively oxidized.
[0341] Regarding "aryl" or "heteroaryl", "mono- or polysubstituted"
are understood to mean the mono- or polysubstitution, e.g. di-,
tri-, 4- or 5-substitution, of one or more hydrogen atoms of the
ring system.
[0342] Particularly preferred are the (hetero)aryl substituents
selected independently of one another from --F, --Cl, --Br, --I,
--CN, --CHO, --CO.sub.2H, --NH.sub.2, --NO.sub.2, --NHR.sub.X,
--N(R.sub.X).sub.2, --N.sup.+(R.sub.X).sub.3, --N.sup.30 l
(R.sub.X).sub.2O.sup.-, --SH, --SR.sub.X, --OH, --OR.sub.X,
--C(.dbd.O)R.sub.X, --CO.sub.2R.sub.X, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NHR.sub.X, --C(.dbd.O)N(R.sub.X).sub.2,
--S(.dbd.O).sub.1-2R.sub.X, --S(.dbd.O).sub.2NH.sub.2, --SO.sub.3H,
.dbd.O or --R.sub.X. Preferred substituents are --F, --Cl, --Br,
--I, --OH, --OC.sub.1-8-alkyl, --O--C(.dbd.O)--C.sub.1-8-alkyl,
--SH, --NH.sub.2, --NHC.sub.1-8-alkyl, --N(C.sub.1-8-alkyl).sub.2,
--C(.dbd.O).sub.1-8-alkyl or --C(.dbd.O)OH. Particularly preferred
substituents are --F, --Cl, --OH, --SH, --NH.sub.2 and
--C(.dbd.O)OH.
[0343] Unless expressly stated otherwise, residues having more than
a single binding partner can be attached in any direction. For
example, the residue "--S--(CH.sub.2)--C(.dbd.O)--" which is
attached to binding partners B.sub.1 and B.sub.2 can be present in
either direction. B.sub.1--S--(CH.sub.2)--C(.dbd.O)--B.sub.2 or
B.sub.1-C(.dbd.O)--(CH.sub.2)--S--B.sub.2.
[0344] Particularly preferred are compounds according to general
formulas A), (B) (C), (D), (E) and (F), respectively, wherein
[0345] R.sub.A0 means --H; R.sub.A1 and R.sub.A2 together with the
carbon atoms to which they are attached form a ring and mean
--O--CH.sub.2--O-- or --O--CH(CH.sub.3)--O--; R.sub.A6 means --H or
--CH.sub.3; R.sub.A7 means --CO.sub.2H; R.sub.A3, R.sub.A4, and
R.sub.A5, respectively independently, mean --OCH.sub.3; or R.sub.A3
means --H; and R.sub.A4 and R.sub.A5 together with the carbon atoms
to which they are attached form a ring and mean --O--CH.sub.2--O--;
and m.sub.A means 0 or 1; [0346] R.sub.B1 means --H or --CH.sub.3;
R.sub.B2 and R.sub.B3 mean CO.sub.2H; X.sub.B1 and X.sub.B2 mean
--O--; m.sub.B means 1 and n.sub.B means 1; [0347] A.sub.C means O
or NR.sub.C3; R.sub.C1 means CH.sub.3 or 4-methoxyphenyl; R.sub.C2
means --H or --CH.sub.2--S--CH.sub.2--(5-methylfuran-2-carboxylic
acid), R.sub.C3 means --(CH.sub.2).sub.2--C(.dbd.O)OH; R.sub.C4
means CO.sub.2H; m.sub.c means 0 or 2; [0348] X.sub.D1 means O or
NR.sub.D3; X.sub.D2 means S; Y.sub.D means --CH.sub.2--,
--X.sub.D2--(CH.sub.2)--C(.dbd.O)--; or
--C(.dbd.O)--(CH.sub.2)--X.sub.D2--; A.sub.D1 means NH or CH;
A.sub.D2 means N or C; A.sub.D3 means N; with the proviso that
A.sub.D1 means CH or NH, while A.sub.D2 means N or C, respectively,
R.sub.D1 means --H or --Cl; R.sub.D2 means --H; R.sub.D3 means H;
R.sub.D4 means CO.sub.2H; [0349] R.sub.E1 and R.sub.E2,
respectively independently, mean --OCH.sub.3; R.sub.E3 and
R.sub.E4, respectively independently, mean --H; R.sub.E5 means
--C(.dbd.O)OH; m.sub.E means 1; and n.sub.E means 1; [0350]
R.sub.F1 and R.sub.F2 mean --CH.sub.3; R.sub.F3 means
--C(CH.sub.3).sub.3; R.sub.F4 means --CO.sub.2H; Y.sub.F means
--CH.sub.2C(.dbd.O)NH--(CH.sub.2).sub.2--; X.sub.F means .dbd.O;
A.sub.F1 and A.sub.F2 mean --O--.
[0351] The compounds according to the invention can be present in
the form of a single stereoisomer or mixture thereof, the free
compounds and/or their physiologically compatible salts and/or
prodrugs and/or solvates.
[0352] The compounds according to the invention be chiral or
achiral, depending on the substitution pattern.
[0353] If the compounds according to the invention are chiral, then
they are preferably present as racemate or a mixture of
stereoisomers or diastereomers or in enriched form of an
enantiomer.
[0354] In a preferred embodiment the enantiomer excess (ee) of the
S-enantiomer amounts to at least 50% ee, more preferred at least
75% ee, more preferred at least 90% ee, most preferred at least 95%
ee, and in particular at least 99% ee. In another preferred
embodiment, the enantiomer excess (ee) of the R-enantiomer amounts
to at least 50% ee, more preferred at least 75% ee, more preferred
at least 90% ee, most preferred at least 95% ee, and in particular
at least 99% ee.
[0355] Suitable methods for separating the enantiomers are known to
the person skilled in the art. Preparative HPLC on chiral
stationary phases and conversion into diastereomeric intermediates
can be given as examples. The conversion into diastereomeric
intermediates can occur, for example, as salt formation by means of
chiral, enantiomer-pure acids. After separation of the
diastereomers thus formed, the salt can then be converted into the
free base or another salt again.
[0356] Unless expressly specified, each reference to the compounds
according to the invention covers all isomers in pure form and
admixture with one another (e.g. stereoisomers, diastereomers,
enantiomers) in any desired mixture ratio.
[0357] Unless expressly specified, each reference to the compounds
according to the invention covers the free compounds (i.e. the
forms that are not present in the form of salt) and all
physiologically compatible salts and all prodrugs.
[0358] For the purposes of the description, physiologically
compatible salts of the compounds according to the invention are
present as salts with anions or acids of the respective compound
with inorganic or organic acids, which are physiologically
compatible--in particular on application in humans and/or
mammals.
[0359] Examples of physiologically compatible salts of specific
acids are salts of: hydrochloric acid, hydrobromic acid, sulphuric
acid, methane sulphonic acid, formic acid, acetic acid, oxalic
acid, succinic acid, malic acid. tartaric acid. mandelic acid,
fumaric acid, lactic acid, citric acid, glutamic acid, saccharinic
acid, monomethyl sebacic acid, 5-oxo-proline, hexane-1-sulphonic
acid, nicotinic acid, 2-, 3- or 4-aminobenzoic acid,
2,4,6-trimethyl benzoic acid, .alpha.-liponic acid, acetylglycine,
acetylsalicylic acid, hippuric acid and/or aspartic acid. The
hydrochloride, citrate and hemicitrate are particularly
preferred.
[0360] Physiologically compatible salts with cations or bases are
salts of the respective compound--as anion with at least one,
preferably inorganic, cation, which are physiologically
compatible--in particular on application in humans and/or mammals.
Particularly preferred are the salts of the alkali and earth alkali
metals, also ammonium salts, but in particular (mono-) or (di-)
sodium, (mono-) or (di-) potassium, magnesium or calcium salts.
[0361] Prodrugs of the compounds according to the invention are
such which are converted in viva to the pharmacologically active
compound. The most common prodrugs are carboxylic acid esters, e.g.
acetats, ethyl esters. For further details and a detailed list of
prodrug concepts that is not to be construed as limiting the scope
of possible prodrugs, reference is made to e.g. J. Rautio, Nat Rev
Drug Discov, 2008, 7, 255-270.
[0362] The compounds according to the invention are defined by
substituents, e.g. by R.sub.A3, R.sub.A4, and R.sub.A5
(substituents of the first generation). which are themselves
possibly substituted (substituents of the second generation).
Depending on the definition, these substituents of the substituents
can themselves be substituted again (substituents of the third
generation). If, for example, R.sub.A3=--R.sub.X, wherein
--R.sub.X=--C.sub.1-8-aliphatic (substituent of the first
generation), then --C.sub.1-8--aliphatic can itself be substituted,
e.g. with --OR.sub.X, wherein R.sub.X=-aryl (substituent of the
second generation). This gives the functional group
--C.sub.1-8-aliphatic--O--aryl. -Aryl can then in turn be
substituted again, e.g. with --Cl (substituent of the third
generation). This then gives overall the functional group
--C.sub.1-8aliphatic--O--aryl--Cl.
[0363] The compounds according, to the invention act, for example,
as inhibitors of the NHR2 and/or RUNX1/ETO-tetramerization which is
associated with different diseases, and therefore they are suitable
as pharmaceutical active substance in a medication.
[0364] Another aspect of the invention relates to the compounds
according to the invention as described above as medicaments.
[0365] Another aspect of the invention relates to pharmaceutical
compositions or pharmaceutical dosage forms comprising the
compounds and/or prodrugs according to the invention as described
above.
[0366] Preferably, the pharmaceutical compositions comprise a
compound according to the invention as described above, and a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier," as used herein, means a non-toxic, inert
solid, semi-solid or liquid filler, diluent, encapsulating material
or formulation auxiliary of any type.
[0367] Some examples of materials which may serve as
pharmaceutically acceptable carriers are sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; cocoa butter and suppository
waxes; oils such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols; such a
propylene glycol; esters such as ethyl oleate and ethyl Iaurate;
agar: buffering agents; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol, and phosphate buffer
solutions, as well as other non-toxic compatible lubricants such as
sodium lauryl sulfate and magnesium stearate, as well as releasing
agents, coloring agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants may also be
present in the composition, according to the judgment of one
skilled in the art of formulations.
[0368] The pharmaceutical compositions may be administered to
subjects (e.g., humans and other mammals) orally, rectally,
parenterally, intravaginally, intracisternally, intraperitoneally,
topically (as by powders, ointments or drops), bucally.
extracorporeally, e.g. by dialysis, or as an oral or nasal spray.
The term "parenterally," as used herein, refers to modes of
administration, including intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous, intraarticular
injection and infusion.
[0369] Pharmaceutical compositions for parenteral injection
comprise pharmaceutically acceptable sterile aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions and sterile
powders for reconstitution into sterile injectable solutions or
dispersions. Examples of suitable aqueous and nonaqueous carriers,
diluents, solvents or vehicles include water, ethanol, polyols
(polyethylene glycol, propylene glycol, glycerol, and the like, and
suitable mixtures thereof), vegetable oils (such as olive oil) and
injectable organic esters such as ethyl oleate, or suitable
mixtures thereof. Suitable fluidity of the composition may be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0370] These compositions may also contain adjuvants, such as
preservative agents, wetting agents, emulsifying agents, and
dispersing agents. Prevention of the action of microorganisms may
be ensured by various antibacterial and antifungal agents, for
example, parabens, phenol, chlorobutanol, sorbic acid, and the
like, it also may be desirable to include isotonic agents, for
example, sugars, sodium chloride and the like. Prolonged absorption
of the injectable pharmaceutical form may be brought about by the
use of agents delaying absorption. for example, aluminum
monostearate and gelatin.
[0371] In some cases, in order to prolong the effect of a drug, it
is often desirable to slow the absorption of the drug from
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. The rate of absorption of the
drug may depend upon its rate of dissolution, which, in turn, may
depend upon crystal size and crystalline form. Alternatively, a
parenterally administered drug form may be administered by
dissolving or suspending the drug in an oil vehicle.
[0372] Suspensions, in addition to the active compounds, may
contain suspending agents, for example, polyoxyethylene sorbitol,
ethoxylated isostearyl alcohols, and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar, tragacanth, and mixtures thereof.
[0373] If desired, and for more effective distribution, the
compounds may be incorporated into slow-release or
targeted-delivery systems such as polymer matrices, liposomes, and
microspheres. They may be sterilized, for example, by filtration
through a bacteria-retaining filter or by incorporation of
sterilizing agents in the form of sterile solid compositions, which
may be dissolved in sterile water or some other sterile injectable
medium immediately before use.
[0374] Injectable depot forms are made by forming microencapsulated
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide, Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release may be controlled. Depot injectable formulations
also are prepared by entrapping the drug in liposomes or
microemulsions which are compatible with body tissues
[0375] The injectable formulations may be sterilized, for example,
by filtration through a bacterial-retaining filter or by
incorporating sterilizing agents in the form of sterile solid
compositions which may be dissolved or dispersed in sterile water
or other sterile injectable medium just prior to use.
[0376] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents,
suspending agents and the like. The sterile injectable preparation
also may be a sterile injectable solution, suspension or emulsion
in a nontoxic, parenterally acceptable diluent or solvent such as a
solution in 1,3-butanediol, Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0377] Solid dosage forms for oral administration include, but are
not limited to, capsules, tablets, pills, powders, and granules. In
such solid dosage forms, one or more compounds is mixed with at
least one inert pharmaceutically acceptable carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and salicylic
acid: b) binders such as carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants
such as glycerol; d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate; e) solution retarding agents such
as paraffin; f) absorption accelerators such as quaternary ammonium
compounds; g) wetting agents such as cetyl alcohol and glycerol
monostearate: h) absorbents such as kaolin and bentonite clay; and
i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets and pills, the dosage
form may also comprise buffering agents.
[0378] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using lactose or
milk sugar as well as high molecular weight polyethylene
glycols.
[0379] The solid dosage forms of tablets, dragees, capsules, pills,
and granules may be prepared with coatings and shells such as
enteric coatings and other coatings well-known in the
pharmaceutical formulating art. They optionally may contain
opacifying agents and also may be of a composition that they
release the active ingredient(s) only, or preferentially. in a
certain part of the intestinal tract in a delayed manner. Examples
of materials useful for delaying release of the active agent may
include polymeric substances and waxes.
[0380] Compositions for rectal or vaginal administration are
preferably suppositories which may be prepared by mixing the
compounds with suitable non-irritating carriers such as cocoa
butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore
melt in the rectum or vaginal cavity and release the active
compound.
[0381] Liquid dosage forms for oral administration may include, but
are not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds. the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures
thereof.
[0382] Besides inert diluents, the oral compositions may also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.
[0383] Suspensions, in addition to the active compounds, may
contain suspending agents, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar, tragacanth, and mixtures thereof.
[0384] If desired, and for more effective distribution, the
compounds may be incorporated into slow-release or
targeted-delivery systems such as polymer matrices, liposomes, and
microspheres. They may be sterilized, for example, by filtration
through a bacteria-retaining filter or by incorporation of
sterilizing agents in the form of sterile solid compositions, which
may be dissolved in sterile water or some other sterile injectable
medium immediately before use.
[0385] Dosage forms for topical or transdermal administration of a
compound include ointments, pastes, creams, lotions, gels, powders,
solutions, sprays, inhalants or patches. A desired compound is
admixed under sterile conditions with a pharmaceutically acceptable
carrier and any needed preservatives or buffers as may be required,
Ophthalmic formulation, eardrops, eye ointments, powders and
solutions are also contemplated as being within the scope of this
disclosure.
[0386] The ointments, pastes, creams and gels may contain, in
addition to an active compound, animal and vegetable fats, oils,
waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[0387] Powders and sprays may contain, in addition to the
compounds, lactose, talc, silicic acid, aluminum hydroxide, calcium
silicates and polyamide powder, or mixtures of these substances.
Sprays additionally may contain customary propellants such as
chlorofluorohydrocarbons.
[0388] Compounds also may be administered in the form of liposomes.
As is known in the art, liposomes are generally derived from
phospholipids or other lipid substances. Liposomes are formed by
mono- or multi-lamellar hydrated liquid crystals that are dispersed
in an aqueous medium. Any non-toxic, physiologically acceptable and
metabolizable lipid capable of forming liposomes may be used. The
present compositions in liposome form may contain, in addition to
the compounds, stabilizers, preservatives, and the like. The
preferred lipids are the natural and synthetic phospholipids and
phosphatidylcholines (lecithins) used separately or together.
Methods to form liposomes are known in the art.
[0389] Dosage forms for topical administration of a compound
according to the invention as described above include powders,
sprays, ointments and inhalants. The active compound is mixed under
sterile conditions with a pharmaceutically acceptable carrier and
any needed preservatives, buffers or propellants. Ophthalmic
formulations, eye ointments, powders and solutions are also
possible. Aqueous liquid compositions may also be useful.
[0390] Another aspect of the invention relates to the compounds
according to the invention as described above for use in the
treatment or the prevention of a disorder or disease that is
associated with NHR2 and/or RUNX1/ETO-tetramerization.
[0391] Another aspect of the invention relates to the use of
inhibitors of NHR2 and/or RUNX1/ETO-tetramerization, which are
preferably non-peptidic and/or which preferably have a molecular
weight of at most 2000 g/mol, more preferably at most 1000 g/mol,
still more preferably at most 750 g/mol and in particular at most
500 g/mol, for the manufacture of a medicament for the treatment or
the prevention of a disorder or disease that is associated with
NHR2 and/or RUNX1/ETO-tetramerization, preferably leukemia, more
preferably myeloid leukemia, in particular acute myeloid
leukemia.
[0392] Another aspect of the invention relates to the use of
inhibitors of NHR2 and/or RUNX1/ETO-tetramerization, which are
preferably non-peptidic and/or which preferably have a molecular
weight of at most 2000 g/mol, more preferably at most 1000 g/mol,
still more preferably at most 750 g/mol and in particular at most
500 g/mol, for the treatment or the prevention of a disorder or
disease that is associated with NHR2 and/or
RUNX1/ETO-tetramerization, preferably leukemia, more preferably
myeloid leukemia, in particular acute myeloid leukemia.
[0393] Another aspect of the invention relates to the use of the
compounds according to the invention as described above for the
manufacture of a medicament for the treatment or the prevention of
a disorder or disease that is associated with NHR2 and/or
RUNX1/ETO-tetramerization,
[0394] Another aspect of the invention relates to a method of
treating or preventing a disorder or disease that is associated
with NHR2 and/or RUNX1/ETO-tetramerization. the method comprising
the administration of a therapeutically effective amount of a
compound or a physiologically acceptable salt and/or a prodrug or a
pharmaceutical composition or a pharmaceutical dosage form
according to the invention as described above to a subject in need
thereof.
[0395] In a preferred embodiment the disorder or disease that is
associated with NHR2 and/or RUNX1/ETO-tetramerization is cancer or
a hematopoietic disease, preferably leukemia.
[0396] Preferably, the leukemia is myeloid leukemia, particularly
preferably the leukemia is selected from the group consisting of
acute myeloid leukemia, promyeloid leukemia, acute promyeloid
leukemia, promyelocytic leukemia, acute promyelocytic leukemia,
megakaryoblastic leukemia and acute megakaryoblastic leukemia.
[0397] In a preferred embodiment, the compounds according to the
invention are for administration once daily. In another preferred
embodiment, the compounds or physiologically acceptable salts
and/or prodrugs or pharmaceutical compositions or pharmaceutical
dosage forms according to the invention are for administration
multiple daily, in particular twice daily or thrice daily, or more
often or continuously infused.
[0398] In a preferred embodiment, the compounds according to the
invention are administered orally. In another preferred embodiment,
the compounds according to the invention are administered
parenterally, in another preferred embodiment, the compounds
according to the invention are administered systemically, locally
or extracorporeally.
[0399] Another aspect of the invention relates to the use of the
compounds according to the invention for measuring the inhibition
of NHR2 and/or RUNX1/ETO-tetramerization in an assay preferably
selected from the group that includes, but is not limited to, ABCD,
EMSA, ELISA and cross-linking assay.
[0400] Preferably, the ABCD assay is an avidin-biotin complex
DNA-binding assay (ABCD) using 5'-biotinylated oligonucleotides
corresponding to the RUNX1 binding sequence within RUNX3 and PU.1
and mutants of these binding sequences. Preferably, the mutant of
these binding sequences is R3mut, which is incapable of RUNX1
binding. Preferably, the ABCD assay is as further described in the
experimental section.
[0401] For the purpose of the specification, RUNX3 is the RUNX3
promoter and PU.1 is the PU.1 promoter. For the purpose of the
specification. R3 is a double-stranded RUNX3 oligonucleotide
corresponding to the RUNX1 binding sequences derived from the RUNX3
promoter sequence that is optionally biotinylated for detection in
assays. The 5'-biotinylated oligonucleotide corresponding to the
RUNX1 binding sequences within RUNX3 (R3) and its mutant incapable
of RUNX1 binding (R3mut) were: R3: 5'-AGG GCC TGG CCT TGT GGT TCT
GTG GTT GAG GGA CCA GGC-3'; R3mut: 5' AGG GCC TGG CCT TGT TAG TCT
GTT AGT GAG GGA CCA GGC. For the purpose of the specification,
RUNX1/ETO-m5 is a mutant RUNX1'ETO protein in which five amino
acids of the NHR2 domain (amino acid 482-548 in RUNX1/ETO) are
mutated to alanine (positions 498, 502, 533, 536 and 540; C.
Wichmann et al., Blood, 2010, 116(4), 602-613) that way interfering
with the formation of the NHR2 and/or RUNX1/ETO
tetramerization.
[0402] Preferably, for the ABCD assay, the R3 or R3mut
oligonucleotide was annealed to the corresponding antisense
unmodified oligonucleotide to generate the RUNX1 binding site, The
5'-biotinylated oligonucleotide within the human PU.1 promoter
region (PU.1) was: 5'-TGG GCC GCT GTG CGG TGC CTG TGG TAA TGG GCT
GT-3'. Biotinylated double-stranded oligonucleotides were mixed
with nuclear extracts from vector transfected 293T cells in binding
buffer (25 mM HEPES, pH 7.5, 50 mM KCl, 1 mM EDTA, pH 8.0, 10 mM
MgCl2, 5% Glycerol, 1 mM DTT) in the presence of 1% NP40 and 100 ng
of salmon sperm DNA and incubated. Streptavidin magnetic beads
(Dynabeads M-280 Streptavidin, Invitrogen) were then added to the
mixture and incubated for one hour. After washing three times, the
bound proteins were eluted from beads in SDS-PAGE sample buffer and
resolved on SDS-PAGE followed by immunoblotting with anti--HA
(Covance), anti-ETO (C-20, Santa Cruz) or anti--Flag (F3165, Sigma,
Germany) antibody.
[0403] Preferably, for electrophoretic mobility shift assays
(EMSA), 293T cells are transfected with vectors for RUNX1/ETO or
RUNX1/ETO-m5, Cell extracts were prepared by lysing transfected
cells in cell lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 0.1%
Triton X-100, 1 mM DTT, 1 mM EDTA, 10% glycerol) followed by
centrifugation to remove insoluble material. Labeling of the probe
and separation by PAGE was performed as described previously (G.
Huang et al, Blood, 2004; 103, 3200-3207) EMSA were carried out
with equal amounts of protein including a high salt buffer (10 mM
Tris pH 7,5, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.4 M NaCl, 20%
Glycerol, 1 mM DTT) and salmon sperm DNA as an unspecific
competitor. A RUNX1-high-affinity oligo (J. A. Mertz et al.,
Journal of Virology, 2001, 75, 2174-2184) was used as a probe
(5'--CAT GGC CCT TTG CGG TTA GTT AC-3'). Preferably, the EMSA assay
is as further described in the experimental section.
[0404] Preferably, for enzyme-linked immunosorbent assays (ELISA),
wells of a 96 well plate were coated with 100 ng streptavidin
(Dianova) in PBS over night at 4.degree. C. A double-stranded
biotinylated RUNX3 oligonucleotide (100 ng, R3=5'-AGG GCC TGG CCT
TGT GGT TCT GTG GTT GAG GGA CCA GGC-3') was bound to streptavidin
in PBS containing 0.1% BSA and 0.05% Tween-20 for 2 h at RT on the
96 well plate. RUNX1-containing proteins were incubated with
synthetic peptides or chemical compounds in binding buffer (25 mM
HEPES, pH 7.5, 50 mM KCl, 1 mM EDTA, pH 8.0, 10 mM MgCl2, 5%
glycerol, 1 mM DTT) in the presence of 1 .mu.g salmon sperm and 1%
IGEPAL CA-630 for 5 h and subsequently added to the
streptavidin-bound R3 oligonucleotide for 15 min on the 96 well
plate. Binding of RUNX1-containing proteins to the R3
oligonucleotide was detected with a primary anti-myc antibody
(9E10, Santa Cruz) and a secondary HRP-coupled antibody (donkey
anti-mouse IgG-HRP, Santa Cruz). ELISA was developed using the Sure
Blue TMB Microwell Peroxidase substrate (KLP). and the reaction was
stopped with sulfuric acid (1 N). Absorption at 450 nm and 650 nm
was measured with the Spectra Max 340. Molecular Devices.
Preferably, the ELISA assay is as further described in the
experimental section.
[0405] Preferably, for the cross-linking assay, purified NHR2
protein or NHR2-containing protein (5 .mu.M) was incubated with the
peptides at 4.degree. C. for 1 h. The BS.sup.3
(Bis[sulfosuccinimidyl] suberate) crosslink-reaction was performed
at a final concentration of 1 mM for 30 min at RT. Tris--HCl (0.05
M, pH 7.4) was added to the reaction mix and incubated for 10 min
to stop the reaction. Protein-oligomerization was analyzed by
western blotting. For protein detection, the membrane was incubated
with a primary anti-myc antibody (9E10, Santa Cruz) and a secondary
HRP-coupled antibody (goat anti-mouse igG-HRP, Santa Cruz).
Preferably, the cross-linking assay is as further described in the
experimental section.
[0406] Another aspect of the invention relates to the use of the
compounds according to the invention as described above with
respect to assays for the diagnosis of diseases associated with the
tetramerization of NHR2 and/or NHR2 containing proteins.
[0407] Yet, another aspect of the invention relates to the use of
the compounds according to the invention as described above with
respect to assays for screening for tetramerization of NHR2 and/or
of NHR2 containing proteins in vitro, ex vivo, in vivo, in mammals
and humans, and the like.
[0408] Yet, another aspect of the invention relates to the use of
the compounds according to the invention as described above as a
reference compound or competing binder/inhibitor in the
applications as described above, i.e. as a searcher,
biotechnological tool or in diagnostics for inhibition of NHR2
and/or RUNX1/ETO-tetramerization.
[0409] The following examples further illustrate the invention but
are not to be construed as limiting its scope:
Structure-Based Identification of Inhibitors of the NHR2
Tetramerization
[0410] An analysis of the previously identified five hot spots
suggested D533, E536, and W540 as the most suitable template motif
(C, Wichmann et al., Blood, 2010). This template motif was used
together with the structural knowledge of the location and
orientation of these residues' side chains in the crystal structure
in a virtual screening (VS) for inhibitors mimicking the hot spot
interactions. Finally, 80 compounds were selected for experimental
testing
[0411] The inhibition of NHR2 tetramerization was measured in vitro
by tetramer-dependent binding of a RUNX1-NHR2 protein construct to
an immobilized oligonucleotide derived from the RUNX3 promoter
sequence in ELISA and ABCD assays, Selectivity for NHR2 was
demonstrated by the reduced inhibition observed when replacing the
NHR2 domain of the construct with the homologous BCR
tetramerization domain (RUNX1-BCR protein). Furthermore, the
influence of the inhibitors on the viability of RUNX1/ETO-dependent
SKNO-1 cells was determined; as a control RUNX1/ETO-independent
U937 cells were used.
[0412] The principle of the biochemical assays for measuring
inhibition of NHR2 tetramerization is illustrated in FIG. 1.
[0413] NHR2 tetramerization and its inhibition was measured in a
purpose-built sandwich ELISA (enzyme-linked immunosorbent assay;
FIG. 1A). [0414] i) The RUNX1--NHR2 tetramer specifically binds to
the biotinylated RUNX3-oligonucleotide (RUNX3-oligo.sup.bio) that
is immobilized on a streptavidine coated surface (SA). Disrupting
the RUNX1-NHR2 tetramer into dimers leads to a release of the
RUNX1-NHR2 protein from the surface. Antibody-conjugated
horseradish peroxidase (HRP) binds to a primary anti-myc antibody
(.alpha.-myc AB) that binds to the immobilized myc-labeled
RUNX1-NHR2 tetramer. HRP catalyzes the conversion of
3,3',5,5'-tetramethylbenzidine (TMB) to
3,3',5,5'-tetramethylbenzidine diimine. The ELISA signal was
quantified photometrically as the difference of absorbance at 650
nm and 450 nm relative to the corresponding difference of
absorbance in the absence of an inhibitor (--). [0415] ii) The
specificity of this ELISA assay was validated by comparing signal
reduction related to tetramer dependent surface binding of the
RUNX1-NHR2 protein (white bars) or the RUNX1-BCR protein (black
bars; both FIG. 1B) under the following conditions, [0416] (i)
presence of the complete reaction mix but no inhibitor; [0417] (ii)
presence of hot spot alanine mutations in the NHR2 domain
(RUNX1-NHR2-m5; N.A. for RUNX1-BCR); [0418] (iii) absence of a
RUNX1-containing protein (no RUNX1 protein); [0419] (iv) absence of
RUNX3-oligo.sup.bio (no RUNX3-oligo.sup.bio), [0420] (v) presence
of a mutated RUNX3-oligo.sup.bio (oligo R3mut.sup.bio); [0421] (vi)
absence of the primary anti-myc antibody (no .alpha.-myc AB);
[0422] (vii) replacement of the anti-myc antibody by a control
antibody (control AB); or (viii) absence of all components from the
reaction mix (empty). [0423] iii) The inhibitory effect of selected
compounds was confirmed with a complementary ABCD (avidin, biotin,
complex, DNA) assay (FIG. 1C) that differs from the ELISA in that
the SA coated surface is replaced by SA magnetic beads for
segregation of bound tetramer followed by SDS-PAGE, immunoblotting,
and signal quantification consistent with the ELISA,
[0424] FIG. 2 illustrates the inhibition of NHR2 tetramerization,
The following conclusions can be drawn: [0425] a) Peptide P1, but
neither P2, P3 nor the unrelated peptide CP inhibit NHR2
tetramerization as shown by the reduction of BS.sup.3 cross-linked
NHR2 oligomers (c=1100 .mu.M); [0426] b) The inhibition by P1 is
dose-dependent; [0427] c) with IC.sub.50.apprxeq.250 .mu.M; [0428]
d) ELISA; and [0429] e) ABCD experiments show that P1, 7.18, and
7.44, but not 7.38, selectively inhibit tetramer-dependent binding
of the RUNX1-NHR2 protein (white bars) to an immobilized RUNX3
oligonucleotide while binding of the RUNX1-BCR protein (black bars)
is not inhibited ([P1]=500 .mu.M and [PPM]=2000 .mu.M in ELISA;
c=1100 .mu.M in ABCD assay); [0430] f) dose-dependent inhibition of
RUNX1-NHR2 tetramerization in the ELISA by P1 (.circle-solid.;
IC.sub.50=460 .mu.M) and 7.44 (.diamond-solid.; IC.sub.50=680
.mu.M) but neither by P3 () nor by 7.38 (.box-solid.); [0431]
standard deviations for n.gtoreq.2; ns: not significant; **:
0.01>p.gtoreq.0.001; ***: p<0,0001 (unpaired t-test). [0432]
iv) The inhibitory effect of selected compounds in the ELISA assay
was demonstrated by comparing signal reduction related to tetramer
dependent surface binding of the RUNX1-NHR2 protein (white bars) or
the RUNX1-BCR protein (black bars; both FIG. 2D) under the
following conditions: [0433] (i) presence of the complete reaction
mix but no inhibitor (31 -); [0434] (ii) absence of
RUNX3-oligo.sup.bio (no oligo); [0435] (iii) presence of a mutated
RUNX3-oligo.sup.bio (mut. oligo); [0436] (iv) presence of the
complete reaction mix and the lamer peptide inhibitor P1 at a final
concentration of 500 .mu.M (P1; sequence given in FIG. 4A) [0437]
(v) presence of the complete reaction mix and the inhibitor 7.18 at
a final concentration of 2000 .mu.M; [0438] (vi) presence of the
complete reaction mix and the inhibitor 7.44 at a final
concentration of 2000 .mu.M. [0439] v) The inhibitory effect of
selected compounds in the ABCD assay was demonstrated by comparing
signal reduction related to tetramer dependent surface binding of
the RUNX1-NHR2 protein (white bars) or the RUNX1-BCR protein (black
bars; both FIG. 2E) under the following conditions; [0440] (i)
presence of the complete reaction mix but no inhibitor (--); [0441]
(ii) absence of RUNX3-oligo.sup.bio (no oligo); [0442] (iii)
presence of a mutated RUNX3-oligo.sup.bio (mut. oligo); [0443] (iv)
presence of the complete reaction mix and the non-inhibitory
reference substance 7.38 at a final concentration of 1100 .mu.M;
[0444] (v) presence of the complete reaction mix and the inhibitor
7.18 at a final concentration of 1100 .mu.M;
[0445] FIG. 4 shows: [0446] a) Sequences of the peptides used in
NHR2 inhibition assays aligned to the wild type NHR2 sequence
(alanine mutations highlighted); corresponding sequence positions
according to RUNX1/ETO numbering are denoted for the NHR2 sequence;
all peptides were C-terminally acetylated and N-terminally
amidated; [0447] b) protein constructs used in ELISA and ABCD
assays; the wild type NHR2 tetramerization domain or the homologous
BCR tetramerization domain (highlighted) are optionally enclosed by
an N-terminal RUNX1 domain and C-terminal c-myc and His6 tags; the
alanine mutations in the RUNX1-NHR2-m5 protein (indicated by the
"A"s above the NHR2 domain) correspond to RUNX1/ETO positions 526,
530, 533, 536, and 540. in accord with the alanine mutations of P2
depicted in a). [0448] vi) The dose-dependency of the inhibitory
effect of selected compounds in the ELISA assay was demonstrated by
measuring the signal reduction related to tetramer dependent
surface binding of the RUNX1-NHR2 protein (FIG. 2F) under the
following conditions: [0449] (i) presence of the complete reaction
mix and the 18mer peptide inhibitor P1 at different concentrations
(.circle-solid.; sequence of P1 given in FIG. 4A); [0450] (ii)
presence of the complete reaction mix and the inhibitor 7.44 at
different concentrations (.diamond-solid.); [0451] (iii) presence
of the complete reaction mix and the non-inhibitory 18mer peptide
P3 at different concentrations (; sequence of P3 given in FIG. 4A);
[0452] (iv) presence of the complete reaction mix and the
non-inhibitory reference substance 7.38 at different concentrations
(.box-solid.); [0453] vii) The inhibitory effect of selected
compounds in the ELISA assay was quantified by means of IC.sub.50
values and the inhibition in the presence of an inhibitor at a
specific concentration relative to the absence of that particular
inhibitor (relative inhibition in %). Each measurement was carried
out in the presence of the complete reaction mix and in the
presence of an inhibitor or reference compound from the group
consisting of P1, P3, 2b, 5.4. 5.8, 5.9, 5.17, 5.18, 7.3, 7.4,
7.18, 7.38, and 7.44 (Table 1 below).
[0454] The effect of the inhibition of NHR2 tetramerization on
cells was measured in a cell survical assay (FIG. 3). It becomes
clear that NHR2 inhibitor 7.44 specifically reduces proliferation
of RUNX1/ETO-dependent cells; SKNO-1 cell or U937 cells were
treated with 1 .mu.M (.box-solid.) or 10 .mu.M (.tangle-solidup.)
of 7.44 or 7.38 or no PRIM (.circle-solid.). The following
conclusions can be drawn: [0455] a) RUNX1/ETO-dependent SKNO-1
cells were severely affected by 7.44: [0456] b) while
RUNX1/ETO-independent U937 cells were unaffected; 7.38, inactive in
vitro, did not affect. SKNO-1 cells; proliferation measured by XTT
assay; standard deviations for n.gtoreq.3. NHR2 dependent SKNO-1
cells and NHR2 independent U937 cells were cultured, Cell
proliferation and viability were measured with the cell
proliferation kit II (XTT, Roche Applied Science), The XTT assay
was performed at days 3, 5, and 7, 3000 cells per well were
cultured in a 96 well plate and daily treated with compounds under
the following conditions: [0457] (i) SKNO-1 cells were daily
treated with 0 .mu.M of compound 7.44 (.circle-solid.; FIG. 3A);
[0458] (ii) SKNO-1 cells were daily treated with 1 .mu.M of
compound 7.44 (.box-solid.; FIG. 3A): [0459] (iii) SKNO-1 cells
were daily treated with 10 .mu.M of compound 7.44
(.tangle-solidup.; FIG. 3A); [0460] (iv) U937 cells were daily
treated with 0 .mu.M of compound 7.44 (.circle-solid.; FIG. 3B);
[0461] (v) U937 cells were daily treated with 1 .mu.M of compound
7.44 (.box-solid.; FIG. 3B); [0462] (vi) U937 cells were daily
treated with 10 .mu.M of compound 7.44 (.tangle-solidup.; FIG. 3B);
[0463] (vii) SKNO-1 cells were daily treated with 0 .mu.M of
compound 7.38 (.circle-solid.; FIG. 3C): [0464] (viii) SKNO-1 cells
were daily treated with 1 .mu.M of compound 7.38 (.box-solid.: FIG.
3C); or [0465] (ix) SKNO-1 cells were daily treated with 10 .mu.M
of compound 7.38 (.tangle-solidup.; FIG. 3C).
[0466] For the purpose of the specification, P1 is an inhibitory
18mer peptide (sequence given in FIG. 4A), P2, P3, and CP are
non-inhibitory 18mer peptides (sequences given in FIG. 4A), and 2b,
5.4, 5.8, 5.9, 5.17, 5.18, 7.3, 7.4, 7.18, 7.38, and 7.44 are
chemical compounds, wherein [0467] 2b is
2-(imidazo[1,2-a]pyridin-2-ylmethoxy)benzoic acid, [0468] 5.4 is
5-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)-2-chlorobenzoic acid;
[0469] 5.8 is
3,3'-(5-(4-methoxyphenyl)-1H-pyrrole-1,2-diyl)dipropanoic acid;
[0470] 5.9 is
5,5'-dimethyl-4,4'-(sulfanediylbis(methyl))-di(furan-2-carboxylic
acid); [0471] 5.17 is
3-(2-(3-tert-butyl-5,9-dimethyl-7-oxo-7H-furo[3,2-g]chromen-6-yl)acetamid-
o)propanoic acid; [0472] 5.18 is
2-(1-((4,7-dimethoxy-1H-indole-2-carboxamido)methyl)cyclohexyl)acetic
acid; [0473] 7.3 is 2-[2-(carboxymethyloxy)-4-methyl-phenoxy]acetic
acid; [0474] 7.4 is 2,2'-(1,3-phenylenebis(oxy))diacetic acid;
[0475] 7.18 is
4-(benzo[d][1,3]dioxol-5-yl)-3-methyl-4-oxo-2-(3,4,5-trimethoxybenzyl)but-
anoic acid; [0476] 7.38 is
(E)-2-(2-carboxyvinyl)-5-methyl-6-oxo-3,6-dihydropyrimidine-4-carboxylic
acid; and [0477] 7.44 is
2,4-di(benzo[d][1,3]dioxol-5-yl)-4-oxobutanoic acid.
[0478] The results are summarized in Table 1 here below;
TABLE-US-00001 TABLE 1 In vitro inhibition of NHR2
tetramerization.sup.[a] ELISA ABCD Comp. IC.sub.50 [.mu.M]
Inhibition [%].sup.[b] Inhibition [%].sup.[c] Number NHR2.sup.[d]
NHR2.sup.[d] BCR.sup.[e] NHR2.sup.[d] BCR.sup.[e] P1 460 42.8 9.2
N.A. N.A. P3 >>2000 N.A. N.A. N.A. N.A. 2b N.A. 83.1 41.5
49.4 11.8 5.4 N.A. 89.7 35.4 63.9 -3.3 5.8 N.A. 70.3 12.0 68.2 56.1
5.9 N.A. 51.7 20.7 52.5 14.6 5.17 N.A. 41.7 15.9 89.5 95.3 5.18
N.A. 80.5 3.9 83.0 52.6 7.3 N.A. 94.4 31.5 57.7 28.5 7.4 N.A. 71.6
10.0 47.4 6.1 7.18 N.A. 47.7 -6.7 80.2 -2.2 7.38 >>2000 4.0
N.A. 20.1 26.1 7.44 680 69.0 24.0 N.A. N.A. .sup.[a]Technical
details of ELISA and ABCD assays are described in Materials and
Methods; .sup.[b]relative inhibition at 1 mM inhibitor
concentration. Inhibition by 2b was measured at 50 .mu.M
concentration; inhibition by 7.3, 7.4, 7.18, and 7.38 was measured
at 2 mM concentration; .sup.[c]relative inhibition at 1 mM
inhibitor concentration; .sup.[d]inhibition of tetramerization of
the RUNX1-NHR2 protein; .sup.[e]inhibition of tetramerization of
the RUNX1-BCR.
[0479] The effect of the inhibition of NHR2 tetramerization was
measured in a mouse model (xenotransplantation of SKNO-1 and K562
cells in SCID mice).
[0480] For the experiment, the SCID- or SCID-gamma chain mouse
model was used. The experiment investigates if the transplantation
of the AML cell line SKNO-1 after treatment with either 7.44 or
7.38 leads to reduced tumor growth. As a control, the CML cell line
K562 treated with 7.44 was used. For this, the cells were treated
in vitro with 25 .mu.M of the respective compound for four days.
Furthermore, untreated SKNO-1 and CML cell lines were used The cell
growth measured daily during this period is shown in FIGS. 5G and
5H. The results show that 7.44 did not influence the growth of K562
cells in vitro but did influence the proliferation of SKNO-1 cells
in vitro. At day 5 the number of living cells was determined, and
5.times.105 SKNO-1 cells or 5.times.105 K562 cells were injected
per transplant. Per mouse, untreated and treated cells were
injected into the left and right flank, respectively.
[0481] At the day of injection, the expression of the surface
marker CD11b, a measure for cell differentiation, was determined
(FIG. 5E) as was the rate of apoptosis (FIG. 5F). Treatment with
7.44 led to an increase of the CD11b expression by 24% in SKNO-1
cells and an increase of the number of early apoptotic cells by
40%. In contrast, treatment of K562 cells with 7.44 did neither
lead to an increase of the CD11b expression nor of the rate of
apoptosis. The control 7.38 only led to an increase of 2% of CD11b
expression in SKNO-1 cells. For determining the tumor volume, the
length and the width of the tumor was measured twice weekly,
starting one week after the transplantation (FIGS. 5A and 5B). For
SKNO-1 cells, treatment with 7.44 led to a strong reduction in
tumor growth in contrast to untreated SKNO-1 cells. Treatment with
7.38 only led to a small reduction, which suggests that the solvent
itself may have a small influence. Finally, treatment of K562 cells
with 7.44 did not show any significant influence on tumor growth.
At the end of the experiment, the tumors were freed and weighed
(FIGS. 5C and 5D). The results show again that 7.44 considerably
reduced tumor growth of SKNO-1 cells.
[0482] In summary, the data show a specific action of 7.44 against
tumor growth of RUNX1/ETO-positive SKNO-1 cells.
[0483] FIG. 5 shows reduced tumor growth of SKNO-1 cells in SCID-gc
mice after treatment with 7.44. [0484] a) SKNO-1 cells were treated
daily for 4 days with 25 .mu.M of the compounds 7.44 or 7.38. On
day 5, 5.times.105 SKNO-1 cells were injected subcutaneously into
the left (untreated cells) and right flank (cells treated with 7.44
or 7.38). One week after injection, the size of the tumor was
measured with a caliper. [0485] b) K562 cells were treated daily
for 4 days with 25 .mu.M of compound 7.44. On day 5, 5.times.105
K562-1 cells were injected subcutaneously into the left (untreated
cells) and right flank (cells treated with 7.44). One week after
injection, the size of the tumor was measured with a caliper.
[0486] c) Freed tumors that grew after xenotransplantation of
treated or untreated SKNO-1 or K562 cells. [0487] d) Tumor weight
of the xenotransplants. [0488] e) FACS analyses of the CD11b
expression of SKNO-1 cells and K562 cells before transplantation.
[0489] f) FACS analyses of annexin V- and 7-AAD-positive cells of
the SKNO-1 and K562 cell lines before transplantation. [0490] g, h)
In parallel to the daily treatment with compounds the live cell
number of SKNO-1 (g) and K562 (h) cells was determined in vitro by
trypan blue.
Materials & Methods
Peptides and Small Molecule Inhibitors
[0491] Peptides were synthesized by GenScript and Dr. Diana Imhof
(University of Bonn). Control peptides were kindly provided by Dr.
Joachim Koch (Georg-Speyer-Haus). Peptides were dissolved in
dH.sub.2O. Compounds were obtained from (Sigma Aldrich), (Enamine),
(Chembridge), (TimTec), (Princeton), (Otava), (National Cancer
institute), (Chemonaut). Compounds were dissolved in DMSO only or
in dH.sub.2O followed by dropwise addition of ammonia solution to a
final concentration of 25%.
Protein Preparation
[0492] For protein expression, chemically competent BL21(DE3) E.
coli (Invitrogen) were used. An overnight pre-culture containing
ampicillin (100 .mu.g ml.sup.-1) and glucose (0.8% w/v) was used
the next day to inoculate a fresh culture at a ratio of 1:10. At an
OD.sub.600 value of 0.7, protein expression was induced by IPTG
(250 .mu.M for the NHR2 protein, 100 .mu.M for the RUNX1-NHR2
protein, and 500 .mu.M for the RUNX1-BCR protein), and the culture
was incubated for 4 h at 37.degree. C. For protein lysis, the
bacterial pellet was resuspended in IMAC buffer (20 mM Tris-HCl,
500 mM NaCl, 10% glycerine, 20 mM imidazole, pH 8.0) in the
presence of a protease inhibitor cocktail (P8849, Sigma-Aldrich).
Cell lysis was performed by the addition of lysozyme (1 mg
ml.sup.-1) and subsequent sonication. For protein purification, the
HisTrap HP column (GE Healthcare) was used.
Cross-Linking Assay
[0493] Purified NHR2 protein (5 .mu.M) was incubated with the
peptides at 4.degree. C. for 1 h. The BS.sup.3
(Bis[sulfosuccinimidyl] suberate) crosslink-reaction was performed
at a final concentration of 1 mM for 30 min at RT. Tris-HCl (0.05
M, pH 7.4) was added to the reaction mix and incubated for 10 min
to stop the reaction. Protein-oligomerization was analyzed by
western blotting. For protein detection, the membrane was incubated
with a primary anti-myc antibody (9E10, Santa Cruz) and a secondary
HRP-coupled antibody (goat anti-mouse IgG-HRP, Santa Cruz).
ELISA Assay
[0494] Wells of a 96 well plate were coated with 100 ng
streptavidin (Dianova) in PBS over night at 4.degree. C. A
double-stranded biotinylated RUNX3 oligonucleotide (100 ng,
R3=5'-AGG GCC TGG CCT TGT GGT TCT GTG GTT GAG GGA CCA GGC-3') was
bound to streptavidin in PBS containing 0.1% BSA and 0.05% Tween-20
for 2 h at RT on the 96 well plate, RUNX1-containing proteins were
incubated with synthetic peptides or chemical compounds in binding
buffer (25 mM HEPES, pH 7.5, 50 mM KCl, 1 mM EDTA, pH 8.0, 10 mM
MgCl, 5% glycerol, 1 mM DTT) in the presence of 1 .mu.g salmon
sperm and 1% IGEPAL CA-630 for 5 h and subsequently added to the
streptavidin-bound R3 oligonucleotide for 15 min on the 96 well
plate. Binding of RUNX1-containing proteins to the R3
oligonucleotide was detected with a primary anti-myc antibody
(9E10, Santa Cruz) and a secondary HRP-coupled antibody (donkey
anti-mouse IgG-HRP, Santa Cruz). ELISA was developed using the Sure
Blue TMB Microwell Peroxidase substrate (KLP), and the reaction was
stopped with sulfuric acid (1 N). Absorption at 450 nm and 650 nm
was measured with the Spectra Max 340, Molecular Devices.
ABCD Assay
[0495] The sequence of the 5'-biotinylated oligonucleotide
corresponding to the RUNX1 binding sequences within RUNX3 (R3) and
its mutant (R3mut) used in this study were: R3=5' AGG GCC TGG CCT
TGT GGT TCT GTG GTT GAG GGA CCA GGC; R3mut=5' AGG GCC TGG CCT TGT
TAG TCT GTT AGT GAG GGA CCA GGC. Oligonucleotides were annealed to
the corresponding unmodified antisense oligonucleotide to generate
the RUNX1 binding site. Purified RUNX1 proteins were pre-incubated
with a compound for 1 h at 4.degree. C. Similarly the biotinylated
double-stranded oligonucleotides were pre-incubated with
streptavidin coated magnetic beads (Dynabeads M-280 Streptavidin,
invitrogen) for 1 h at 4.degree. C. Protein/compound samples and
beads with bound oligonucleotide were incubated in binding buffer
(25 mM HEPES, pH 7.5, 50 mM KCl, 1 mM EDTA, pH 8.0, 10 mM
MgCl.sub.2, 5% glycerol, 1 mM DTT) in the presence of 2% IGEPAL,
CA-630, 1 .mu.g of salmon sperm, and 0.1% BSA for 1% h. After
washing three times, the bound proteins were eluted from beads in
SDS-PAGE sample buffer and resolved on SDS-PAGE followed by
immunoblotting with a primary anti-myc (9E10, Santa Cruz) antibody
and a secondary HRP-coupled antibody (goat anti-mouse IgG--HRP,
Santa Cruz).
Cell Survival Assays
[0496] SKNO-1 cells were cultured in RPMI 1640+10% FCS and 7 ng
ml.sup.-1 human GM-CSF. U937 cells were cultured in RPMI 1640+10%
FCS. Cell proliferation and viability were measured with the cell
proliferation kit II (XTT, Roche Applied Science). 3000 cells per
well were cultured in a 96 well plate and daily treated with
compounds at different concentrations. The XTT assay was performed
at days 3, 5, and 7.
Sequence CWU 1
1
8139DNAunknownRUNX1 binding sequence (R3) 1agggcctggc cttgtggttc
tgtggttgag ggaccaggc 39239DNAunknownmutant of RUNX1 binding
sequence incapable of RUNX1 binding (R3mut) 2agggcctggc cttgttagtc
tgttagtgag ggaccaggc 39335DNAhuman 3tgggccgctg tgcggtgcct
gtggtaatgg gctgt 35423DNAartificialRUNX1-high-affinity oligo
4catggccctt tgcggttagt tac 23518PRTunknownwild type NHR2 sequence
(P1) 5Val Leu Arg Arg Cys Gln Glu Ala Asp Arg Glu Glu Leu Asn Tyr
Trp1 5 10 15Ile Arg618PRTartificialmutant P2 6Val Leu Arg Arg Cys
Gln Glu Ala Ala Arg Glu Ala Leu Asn Tyr Ala1 5 10 15Ile
Arg718PRTartificialmutant P3 7Val Ala Arg Arg Cys Ala Glu Ala Ala
Arg Glu Ala Leu Asn Tyr Ala1 5 10 15Ile Arg818PRTartificialmutant
CP 8Val Leu Gln Glu Leu Gln Arg Leu Glu Ser Arg Leu Gln Pro Phe
Leu1 5 10 15Gln Arg
* * * * *